Please select what you would like included for printing: Copy the text below and then paste that into your favorite email application Funeral service will be held at 11 a.m., on Friday, March 28, 2025 at Hosanna Lutheran Church, Mankato. Visitation will be from 9-11 a.m. prior to the service at the church. Burial will be at East Lutheran Cemetery, rural Delavan. Mankato Mortuary is in charge of arrangements. Livestream of the service will be available at: https://boxcast.tv/view/dean-hoechst-funeral-ka6cso2kgejeuzp2cazh 1938 at home to Palmer and Bernita (Kunz) Hoechst Dean graduated from Minnesota Lake High School in 1956 1964 Dean was united in marriage to Lois Pieper at St Pauls United Church of Christ in rural Minnesota Lake and later raised his family and farmed near Delavan primarily driving for Nordaas American Homes Dean and Lois wintered for many years in Mesa and were fortunate enough to travel to many different states entertaining with friends and working on the farm especially his grandchildren and great-grandson Chad (Shannon) Hoechst of Minnesota Lake; grandchildren Breckin Hoechst and Delaney Hoechst; great-grandchildren Marilyn Rauenhorst as well as several nieces and nephews The family expresses their sincere gratitude to the Mapleton Community Home and Ecumen Hospice for the wonderful care given to dad Enter your phone number above to have directions sent via text This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply Service map data © OpenStreetMap contributors Metrics details Malassezia is a lipophilic commensal yeast that resides mainly on the mammalian skin and is also found to associate with the internal organs Dysbiosis of Malassezia is related to several diseases and often escapes detection as it is difficult to culture and maintain Malassezia cell wall differs from other budding yeasts like S cerevisiae due to the difference in the lipid content and is difficult to transform we present a methodology to stain Malassezia's nucleus and perform cell cycle studies staining presents a challenge due to its exceptionally thick cell wall with high lipid content Our novel methodology addresses this challenge and enables the staining of the Malassezia nucleus with a low background This would allow researchers to visualize the overall nuclear health specifically nuclear morphology and analyze DNA content By employing DNA-specific dyes like DAPI or Hoechst and using PI we can differentiate cells in distinct cell cycle phases using techniques like flow cytometry This novel staining methodology unlocks the door for in-depth cell cycle analysis in Malassezia which has challenged us through ages being refractory to genetic manipulations paving the way for a deeper understanding of this commensal fungus and its potential role in human health 25 mg of DAPI (Sigma) was dissolved in 1 ml of molecular-grade water and the stock solution was stored at − 20 °C 40 μl from the 25 mg/ml DAPI stock solution was dissolved in 960 μl of molecular-grade water to make a working solution (1 mg/ml) This working solution was stored at 4 °C 25 mg of Hoechst (Sigma) was dissolved in 1 ml of molecular-grade water and the stock solution was stored at − 20 °C 40 μl from the 25 mg/ml Hoechst stock solution was dissolved in 960 μl of molecular-grade water to make a working solution (1 mg/ml) 10 mg of Zymolyase powder (MP Biomedicals) was dissolved in 1 ml of 0.1 M sodium phosphate solution and the stock solution (10 mg/ml) was stored at 4 °C For 0.1 M Sodium phosphate solution 339.4 mg of sodium phosphate monobasic (HiMedia) and 2.021 g of sodium phosphate dibasic (HiMedia) were added with 80 ml of Milli-Q water were mixed with 80 ml of Milli-Q water The Triton X-100 solution was prepared by dissolving Triton X-100 in Milli-Q-Water at a 1:9 ratio 1 mg of RNase powder was added to 1 ml of freshly prepared 1X PBS solution to prepare a stock solution (1 mg/ml) which was kept at − 20 °C 100 μl from the 1 mg/ml RNase A stock was added to 900 μl of 1X PBS (100 μg/ml working solution) and stored at 4 °C 1 mg of Propidium Iodide powder was dissolved in 1 ml of molecular-grade water 50 μl from the 1 mg/ml PI stock was dissolved in 950 μl of molecular-grade water (50 μg/ml working solution) The Cell Permeabilizing Solution (CP Solution) 545 μl used per sample comprised 40 μl of Zymolyase (10 mg/ml) 10 mg of Nocodazole was mixed in 1 ml of DMSO to make a 10 mg/ml stock and stored at 4 °C until use 25 mg of chloramphenicol is mixed in 1 ml of 95% ethanol to make a 25 mg/ml stock solution Yeast cultures were grown in Sabouraud Dextrose (SD) media in an Incubator Shaker (Innova 44R Optical density was measured using a Digital photo colorimeter (Sky Technologies) Fluorescent images were captured using the fluorescence microscope (Oxion fluorescence Cell cycle analysis was done using the Flow cytometer (DxFlex Beckman Coulter) and for data acquisition CytExpert (v2.6) was used For flow cytometry data analysis Kaluza analysis (v1.6) was used and Adobe Illustrator (2023) was used for designing graphics and artwork Comparison of nuclear staining profiles of yeasts using traditional methodology. Nucleus as observed in (a) S. cerevisiae and (b) M. pachydermatis using the stains DAPI and Hoechst. Critical elements contributing to the development of the novel methodology. DAPI staining using (a) Zymolyase, (b) Triton X-100 solution, and (c) both Zymolyase and Triton X-100 (CP solution). Treatment of the cells was carried out for 0, 1, and 2 h. The yellow highlighted box indicates the obtained distinct nucleus. Nuclear staining profiles of Malassezia using the novel methodology. Nuclear staining protocol for M. pachydermatis using (a) DAPI and (b) Hoechst staining. Microscopic images and FACS profiles illustrating cell cycle arrest induced by nocodazole in (a) Saccharomyces cerevisiae (asynchronous cells without nocodazole and cells treated with nocodazole for 0 h and 3 h) and (b) Malassezia pachydermatis (asynchronous cells without nocodazole and cells treated with nocodazole for 0 h Black arrows indicate large budded cells resulting from nocodazole-induced arrest we have provided a DNA staining methodology specifically tailored for fungi of the Malassezia genus which is a challenging task due to their unique structural barriers we employed a combination of enzymatic and chemical treatments to partially permeabilize their thick cell walls without significantly compromising their viability This allowed us to successfully introduce DAPI and Hoechst dyes into the cells enabling precise staining of the nucleus- an achievement not previously attained In addition to this novel staining methodology we undertook a cell cycle analysis of Malassezia A brief timing steps associated with the protocol is presented in Supplementary Fig. 4 and we have also provided a troubleshooting guide for the users in Supplementary Table-1 Utilizing the cell cycle arresting agent Nocodazole we were able to halt cells at the G2/M phase as confirmed by both microscopy and FACS analysis This pioneering work provides researchers with a direct visualization of the Malassezia nucleus and its cell cycle facilitating the identification of its various phases By combining this method with DNA damage-inducing agents we can gain valuable insights into how Malassezia responds to genotoxic stress and repairs DNA thereby opening avenues for novel antifungal strategies Understanding the regulatory mechanisms governing the Malassezia cell cycle would lay the groundwork for unravelling the molecular events that drive cell division This knowledge offers opportunities to explore molecular pathways and signalling cascades involved in cell cycle transitions potentially revealing therapeutic targets for the regulation of Malassezia and related fungi All data generated or analyzed during this study are included in this published article (and its Supplementary Information files) Mittag, H. Fine structural investigation of Malassezia furfur: I Size and shape of the yeast cells and a consideration of their ploidy. Mycoses 37, 393–399. https://doi.org/10.1111/j.1439-0507.1994.tb00388.x (1994) Shetty, M., Naik, B. & Prasad Das, S. Rice bran oil supports robust growth of the commensal fungus Malassezia. The Microbe 3, 100080. https://doi.org/10.1016/j.microb.2024.100080 (2024) P.N. Brito, L.C. Paulino, Phylogenetic analysis reveals unexplored fungal diversity on skin, (2023) 2023.11.22.568368. https://doi.org/10.1101/2023.11.22.568368 Billamboz, M. & Jawhara, S. Anti-malassezia drug candidates based on virulence factors of malassezia-associated diseases. Microorganisms 11, 2599. https://doi.org/10.3390/microorganisms11102599 (2023) Ianiri, G., Dagotto, G., Sun, S. & Heitman, J. Advancing functional genetics through agrobacterium-mediated insertional mutagenesis and CRISPR/Cas9 in the commensal and pathogenic yeast Malassezia. Genetics 212, 1163–1179. https://doi.org/10.1534/genetics.119.302329 (2019) Theelen, B. et al. Malassezia ecology, pathophysiology, and treatment. Med. Mycol. 56, S10–S25. https://doi.org/10.1093/mmy/myx134 (2018) Iliev, I. D. & Leonardi, I. Fungal dysbiosis: immunity and interactions at mucosal barriers. Nat. Rev. Immunol 17, 635–646. https://doi.org/10.1038/nri.2017.55 (2017) Naik, B., Ahmed, S. M. Q., Laha, S. & Das, S. P. Genetic susceptibility to fungal infections and links to human ancestry. Front. Genet. 12, 709315. https://doi.org/10.3389/fgene.2021.709315 (2021) Gaitanis, G., Magiatis, P., Hantschke, M., Bassukas, I. D. & Velegraki, A. The malassezia genus in skin and systemic diseases. Clin. Microbiol. Rev. 25, 106–141. https://doi.org/10.1128/CMR.00021-11 (2012) Saunte, D. M. L., Gaitanis, G. & Hay, R. J. Malassezia-associated skin diseases, the use of diagnostics and treatment. Front. Cell Infect. Microbiol. https://doi.org/10.3389/fcimb.2020.00112 (2020) Malassezia beyond tinea versicolor: the newest wolf in sheep’s clothing pathogen? Abdillah, A. & Ranque, S. Chronic diseases associated with malassezia yeast. J. Fungi (Basel) 7, 855. https://doi.org/10.3390/jof7100855 (2021) Aykut, B. et al. The fungal mycobiome promotes pancreatic oncogenesis via MBL activation. Nature 574, 264–267. https://doi.org/10.1038/s41586-019-1608-2 (2019) Das, S. P., Ahmed, S. M. Q., Naik, B., Laha, S. & Bejai, V. The human fungal pathogen Malassezia and its role in cancer. Fungal Biol. Rev. 38, 9–24. https://doi.org/10.1016/j.fbr.2021.08.002 (2021) Naik, B., Sasikumar, J. & Das, S. P. Fungal coexistence in the skin mycobiome: a study involving Malassezia, Candida, and Rhodotorula. AMB Expr 14, 26. https://doi.org/10.1186/s13568-024-01674-8 (2024) Jayasudha, R. et al. Implicating dysbiosis of the gut fungal microbiome in uveitis, an inflammatory disease of the eye. Investig. Ophthalmol. Vis. Sci. 60, 1384–1393. https://doi.org/10.1167/iovs.18-26426 (2019) Gu, X., Cheng, X., Zhang, J. & She, W. Identification of the fungal community in otomycosis by internal transcribed spacer sequencing. Front. Microbiol. https://doi.org/10.3389/fmicb.2022.820423 (2022) Cleland, E. J. et al. The fungal microbiome in chronic rhinosinusitis: richness, diversity, postoperative changes and patient outcomes. Int. Forum Allergy Rhinol. 4, 259–265. https://doi.org/10.1002/alr.21297 (2014) Dupuy, A. K. et al. Redefining the human oral mycobiome with improved practices in amplicon-based taxonomy: discovery of malassezia as a prominent commensal. PLOS ONE 9, e90899. https://doi.org/10.1371/journal.pone.0090899 (2014) Hammad, D. B. M., Liyanapathirana, V. & Tonge, D. P. Molecular characterisation of the synovial fluid microbiome in rheumatoid arthritis patients and healthy control subjects. PLoS One 14, e0225110. https://doi.org/10.1371/journal.pone.0225110 (2019) Pisa, D., Alonso, R. & Carrasco, L. Parkinson’s disease: a comprehensive analysis of fungi and bacteria in brain tissue. Int. J. Biol. Sci. 16, 1135–1152. https://doi.org/10.7150/ijbs.42257 (2020) Ertam, I., Aytimur, D. & Alper, S. Malassezia furfur onychomycosis in an immunosuppressed liver transplant recipient. IJDVL 73, 425. https://doi.org/10.4103/0378-6323.37067 (2007) Kamamoto, C. S. L. et al. Cutaneous fungal microbiome: Malassezia yeasts in seborrheic dermatitis scalp in a randomized, comparative and therapeutic trial. Dermato-Endocrinol. https://doi.org/10.1080/19381980.2017.1361573 (2017) Shek, Y. H., Tucker, M. C., Viciana, A. L., Manz, H. J. & Connor, D. H. Malassezia furfur—Disseminated infection in premature infants. Am. J. Clin. Pathol. 92, 595–603. https://doi.org/10.1093/ajcp/92.5.595 (1989) Rosales, C. M., Jackson, M. A. & Zwick, D. Malassezia furfur meningitis associated with total parenteral nutrition subdural effusion. Pediatr. Dev. Pathol. 7, 86–90. https://doi.org/10.1007/s10024-003-4030-5 (2004) Olar, A. et al. Central nervous system involvement by malassezia restricta, an unusual fungal pathogen: a report of two cases (P01245). Neurology 78, P01.245. https://doi.org/10.1212/WNL.78.1_supplement.P01.245 (2012) Invasive fungal infection of the central nervous system caused by rare yeast pathogen Malassezia spp in patients with acute leukemia: case reports and literature review Alonso, R., Pisa, D., Aguado, B. & Carrasco, L. Identification of fungal species in brain tissue from Alzheimer’s disease by next-generation sequencing. J. Alzheimers Dis. 58, 55–67. https://doi.org/10.3233/JAD-170058 (2017) Alonso, R., Fernández-Fernández, A. M., Pisa, D. & Carrasco, L. Multiple sclerosis and mixed microbial infections. Direct identification of fungi and bacteria in nervous tissue. Neurobiol. Dis. 117, 42–61. https://doi.org/10.1016/j.nbd.2018.05.022 (2018) Alonso, R., Pisa, D., Fernández-Fernández, A. M., Rábano, A. & Carrasco, L. Fungal infection in neural tissue of patients with amyotrophic lateral sclerosis. Neurobiol. Dis. 108, 249–260. https://doi.org/10.1016/j.nbd.2017.09.001 (2017) Alonso, R., Pisa, D. & Carrasco, L. Brain microbiota in Huntington’s disease patients. Front. Microbiol. https://doi.org/10.3389/fmicb.2019.02622 (2019) Naik, B., Sasikumar, J. & Das, S. P. From skin and gut to the brain: The infectious journey of the human commensal fungus Malassezia and its neurological consequences. Mol. Neurobiol. https://doi.org/10.1007/s12035-024-04270-w (2024) Li, J. et al. Presence of Malassezia hyphae is correlated with pathogenesis of seborrheic dermatitis, microbiology. Spectrum 10, e01169-e1221. https://doi.org/10.1128/spectrum.01169-21 (2022) Bucevičius, J., Lukinavičius, G. & Gerasimaitė, R. The use of hoechst dyes for DNA staining and beyond. Chemosensors 6, 18. https://doi.org/10.3390/chemosensors6020018 (2018) Kapuscinski, J. DAPI: a DNA-specific fluorescent probe. Biotechnol. Histochem. 70, 220–233. https://doi.org/10.3109/10520299509108199 (1995) Reddy, B. S. P., Sondhi, S. M. & Lown, J. W. Synthetic DNA minor groove-binding drugs☆. Pharmacol. Therapeut. 84, 1–111. https://doi.org/10.1016/S0163-7258(99)00021-2 (1999) Specific binding of Hoechst 33258 to site 1 thymidylate synthase mRNA Siljak-Yakovlev, S., Pustahija, F., Vicic, V. & Robin, O. Molecular cytogenetics (FISH and fluorochrome banding): Resolving species relationships and genome organization. Methods Mol. Biol. 1115, 309–323. https://doi.org/10.1007/978-1-62703-767-9_15 (2014) König, K., Riemann, I., Fischer, P. & Halbhuber, K. J. Multiplex FISH and three-dimensional DNA imaging with near infrared femtosecond laser pulses. Histochem Cell Biol. 114, 337–345. https://doi.org/10.1007/s004180000185 (2000) Chapter 2 assays of cell viability: Discrimination of cells dying by apoptosis In Methods in Cell Biology (eds Darzynkiewicz Atale, N., Gupta, S., Yadav, U. C. S. & Rani, V. Cell-death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques. J. Microsc. 255, 7–19. https://doi.org/10.1111/jmi.12133 (2014) Cecchini, M. J., Amiri, M. & Dick, F. A. Analysis of cell cycle position in mammalian cells. JoVE https://doi.org/10.3791/3491 (2012) L. Ding, J. Cao, W. Lin, H. Chen, X. Xiong, H. Ao, M. Yu, J. Lin, Q. Cui, The roles of cyclin-dependent kinases in cell-cycle progression and therapeutic strategies in human breast cancer. Int. J. Mol. Sci. 21 (2020). https://doi.org/10.3390/ijms21061960 Blajeski, A. L., Phan, V. A., Kottke, T. J. & Kaufmann, S. H. G1 and G2 cell-cycle arrest following microtubule depolymerization in human breast cancer cells. J. Clin. Invest. 110, 91–99. https://doi.org/10.1172/JCI13275 (2002) Endo, K., Mizuguchi, M., Harata, A., Itoh, G. & Tanaka, K. Nocodazole induces mitotic cell death with apoptotic-like features in Saccharomyces cerevisiae. FEBS Lett. 584, 2387–2392. https://doi.org/10.1016/j.febslet.2010.04.029 (2010) G.M. Cooper, Microtubules, in: The Cell: A Molecular Approach. 2nd Edition, Sinauer Associates, 2000. https://www.ncbi.nlm.nih.gov/books/NBK9932/ (accessed February 21 Laha, S., Das, S. P., Hajra, S., Sau, S. & Sinha, P. The budding yeast protein Chl1p is required to preserve genome integrity upon DNA damage in S-phase. Nucleic Acids Res. 34, 5880–5891. https://doi.org/10.1093/nar/gkl749 (2006) [62] Visualization of yeast mitochondrial DNA with the fluorescent stain “DAPI” Naik, B., Sasikumar, J. & Das, S. P. Fungal coexistence in the skin mycobiome: a study involving Malassezia Candida, and Rhodotorula. AMB Exp. 14, 26. https://doi.org/10.1186/s13568-024-01674-8 (2024) Cho, Y.-J., Yang, J., Shin, S.Y., Kim, H.K., Rintarhat, P., Park, M., Sung, M., Lagree, K., Underhill, D.M., Lee, D.-W., Choi, C.H., Yang, C.-S., Jung, W.H. Live Malassezia strains isolated from the mucosa of patients with ulcerative colitis, (2023) https://doi.org/10.1101/2023.11.28.569113 Kashaf, S. S. et al. Integrating cultivation and metagenomics for a multi-kingdom view of skin microbiome diversity and functions. Nat. Microbiol. 7, 169–179. https://doi.org/10.1038/s41564-021-01011-w (2022) Amberg, D. C., Burke, D. J. & Strathern, J. N. Yeast vital stains: DAPI stain of nuclear and mitochondrial DNA. Cold Spring Harb Protoc https://doi.org/10.1101/pdb.prot4163 (2006) A. Cone, Yeast DAPI Staining, (2016). https://www.protocols.io/view/Yeast-DAPI-Staining-eigbcbw (accessed January 30 Sazer, S. & Sherwood, S. W. Mitochondrial growth and DNA synthesis occur in the absence of nuclear DNA replication in fission yeast. J. Cell Sci. 97, 509–516. https://doi.org/10.1242/jcs.97.3.509 (1990) Favilla, R., Stecconi, G., Cavatorta, P., Sartor, G. & Mazzini, A. The interaction of DAPI with phospholipid vesicles and micelles. Biophys. Chem. 46, 217–226. https://doi.org/10.1016/0301-4622(93)80015-B (1993) A.M. Celis Ramirez, Unraveling lipid metabolism in lipid-dependent pathogenic Malassezia yeasts, (2017). https://dspace.library.uu.nl/handle/1874/356776 (accessed September 5 Lyticase: Endoglucanase and protease activities that act together in yeast cell lysis Nair, A. & Manohar, S. M. A flow cytometric journey into cell cycle analysis. Bioanalysis 13, 1627–1644. https://doi.org/10.4155/bio-2021-0071 (2021) Rosebrock, A. P. Analysis of the budding yeast cell cycle by flow cytometry. Cold Spring Harb Protoc https://doi.org/10.1101/pdb.prot088740 (2017) S.K. Patel, S.R. Sahu, N. Acharya, Cell cycle analysis of candida Albicans by flow cytometry, Bio. Protoc. 13 (2023) e4848. https://doi.org/10.21769/BioProtoc.4848 Santangelo, G., Navari, S. & Bruno, P. A highly specific microtubule depolymerizing agent (nocodazole) affecting meiosis activation in the protozoan Blepharisma japonicum (Ciliata, Heterotrichida), Italian. J. Zool. 64, 227–234. https://doi.org/10.1080/11250009709356201 (1997) Suomalainen, H. & Nurminen, T. The lipid composition of cell wall and plasma membrane of baker’s yeast. Chem. Phys. Lipids 4, 247–256. https://doi.org/10.1016/0009-3084(70)90026-5 (1970) Download references The authors would like to acknowledge funding from the Indian Council of Medical Research (ICMR) in New Delhi SPD is supported by the DHR Grant GIA/2019/000620/PRCGIA/2020-ECD II JS is supported by a junior research fellowship from Yenepoya (Deemed to be University) The authors would like to thank Yenepoya Research Centre for the laboratory facilities and the Department of Oncopathology Yenepoya (Deemed to be University) for the FACS Jayaprakash Sasikumar, Suparna Laha, Bharati Naik & Shankar Prasad Das and providing critical suggestions and artwork BN helped in the initial standardization and review of the manuscript The authors declare no competing interests Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-024-69024-z Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science MN to Lester and Violet (Schnoor) Pieper. She graduated from Minnesota Lake High School in 1961. On September 12 Lois was united in marriage to Dean Hoechst at St Paul’s United Church of Christ in rural Minnesota Lake. Lois worked for Kato Engineering and MET in Winnebago. She also helped Dean with farming. Lois volunteered her time at MRCI. Lois and Dean wintered for many years in Mesa AZ. She enjoyed playing bridge and cooking and entertaining. She loved time spent with her family Marilyn Rauenhorst and Diane Sharits; brothers-in-law Dale (Linda) Hoechst and Duane Hoechst; and several nieces and nephews. She was preceded in death by her parents; brother Deanna Hoechst; and father-in-law and mother-in-law KARACHI: The Competition Commission of Pakistan (CCP) has granted approval for Hoechst Pakistan Limited to acquire trademarks for the pharmaceutical product Claforan from Sanofi Aventis Deutschland GMBH through a trademark assignment agreement the CCP identified the relevant market as ‘systemic antibiotics’ and classified the transaction as a horizontal merger Hoechst Pakistan’s market share is expected to remain stable as the company has been producing Claforan under licence from Sanofi Aventis this acquisition will not disrupt the market balance but will strategically enhance Hoechst’s position The CCP concluded that the acquisition would not result in market dominance authorising the transaction under Section 31(1)(d)(i) of the Competition Act This approval ensures that competitive stability within Pakistan’s systemic antibiotics market is maintained is a prominent manufacturer and distributor within the European pharmaceutical market The transfer of Claforan’s trademarks to Hoechst positions the latter as a significant player in Pakistan’s pharmaceutical sector Development and Special Initiatives Professor Ahsan Iqbal This representational image shows gold bars — AFP/FileKARACHI: Gold prices fell by Rs2,300 per tola on Saturday in.. A man busy in preparation of handmade carpets with the help of traditional machine to earn his livelihood for support.. —Reuters/FileWASHINGTON: Hawaii lawmakers have passed a “first of its kind” bill that.. —AFP/FileTOKYO: Tokyo’s envoy for US tariff talks said Saturday after a second round of.. Copyright © 2025. The News International, All Rights Reserved | Contact Us | Authors Metrics details Understanding the dynamic interactions of ligands to DNA is important in DNA-based nanotechnologies By structurally tracking the dissociation of Hoechst 33258-bound DNA (d(CGCAAATTTGCG)2) complex (H-DNA) with T-jump 2D-IR spectroscopy the ligand is found to strongly disturb the stability of the three C:G base pairs adjacent to A:T the binding site with the broken base pairs being more than triple at 100 ns The strong stabilization effect of the ligand on DNA duplex makes this observation quite striking which dramatically increases the melting temperature and dissociation time MD simulations demonstrate an important role of hydration water and counter cations in maintaining the separation of terminal base pairs The hydrogen bonds between the ligand and thymine carbonyls are crucial in stabilizing H-DNA whose breaking signal appearing prior to the complete dissociation Thermodynamic analysis informs us that H-DNA association is a concerted process where H cooperates with DNA single strands in forming H-DNA the dissociation of central A:T pairs in H-DNA is initiated by breaking the hydrogen bonds between H and thymine carbonyls rather than the fraying of terminal base pairs Such dynamical distributions can be a concern in the applications of small ligands as sensors detecting the local environment of bimolecular a Temperature-ramp FTIR spectra from 5 to 100 °C b 2D-IR surfaces of DNA and H-DNA measured at 70 °C with parallel polarization c The difference spectra between 85 and 70 °C surfaces d Melting curve obtained with SVD analysis of the FTIR spectra for 1 mM DNA and 1 mM H-DNA (circles) and normalized temperature-dependent fluorescence intensity at 446 nm for 200 nM and 2 μM H-DNA (diamonds) The fitted curves obtained from a two-state model are shown with black solid lines e Transient 2D-IR spectra of native DNA at 500 ns and 1 ms and H-DNA spectra at 500 ns and 4 ms measured at Ti = 70 °C and Tf = 85 °C with parallel polarization The spectral feature associated with the loss of A:T base pairs is outlined with a yellow box consisting of three dominant doublet contours on the diagonal representing intensity gain upon DNA melting Both the A ring mode at 1622 cm−1 and the G ring modes at 1540–1580 cm−1 increase in intensity with the loss of base-pair stacking The melting curves of H-DNA show clear concentration dependence with their inflection point ranging from ~50 to ~100 °C The dissociation of native DNA is a simple two-state transition (D ⇄ 2S) involving the dissociation of the duplex (D) into two single strands (S) which can be described with a dissociation constant Kd the H-DNA dissociation is a more complicated process mechanistically due to the three binding partners involved Instead we find that the dissociation of H-DNA is better characterized as a two-state concerted dissociation into its three constituents (DH ⇄ H + 2S) in this range using a Gibbs free energy change for the dissociation process we find that 62% G:C and 32% A:T base pairs in the native DNA duplex break by 500 ns all G:C base pairs have dissociated by 500 ns but no disruption of A:T pairing is distinguishable These observations illustrate how binding of H to the central AAATTT tracts stabilizes the duplex core to dissociation but at the cost of destabilizing and rapidly fraying the G:C termini d Representative t-HDVE spectra at specified delays e Single-frequency kinetic traces on a log timescale representing the dissociation of A:T (A = 1607 cm−1) The amplitude of the R traces is rescaled by a factor of 0.5 Frequency slices are indicated by the color-coded lines in (c) and (f) The positive and negative amplitudes are shown in orange and purple Shaded areas indicate the relaxation kinetics concurrent with the temperature re-equilibration The T-jump signal change is rescaled by the maximum spectral intensity at equilibrium described by ${\Delta}S\prime (t) = {\Delta}S(t)/\max \left( {S_{{\mathrm{eq}}}(T_{\mathrm{i}})} \right)$ The dsDNA-to-ssDNA transition can be characterized by the dissociation of A:T pairs which is observed as paired positive/negative features centered at 1606/1627 cm−1 in the rate distribution plot Note that the spectral features reflecting the loss of G:C pairs are also observed in the same timescale indicating that the remaining intact G:C and A:T base pairs dissociate concertedly The average observed rate is ~105 s−1 for native DNA but is slowed dramatically down to ~2 × 103 s−1 in the presence of H a Average observed rate (λobs) calculated from the amplitude-weighted mean across the maximum. Error bars represent the standard deviation of the averaged rates. b Calculated association (ka) and dissociation (kd) rate constants of DNA (circles) and H-DNA (triangles) and their fits from Eq. (2) *The unit of ka is M−1 s−1 in the DNA association d Free energy barriers of association (${\Delta}G_{\mathrm{a}}^\dagger$) and dissociation (${\Delta}G_{\mathrm{d}}^\dagger$) of DNA and H-DNA and the relaxation rate for this process derived in the Supplementary Eq (S23): $\lambda _{{\mathrm{obs}}} = 3\left[ S \right]_{{\mathrm{eq}}}^2k_{\mathrm{a}} + k_{\mathrm{d}}$ where [S]eq is expressed with Kd and CD in the Supplementary Eq To quantify the barriers for the dissociation and association reactions we analyzed the temperature-dependent kinetics using the Eyring equation where i = a or d for the association and dissociation process where h is Planck’s constant and kB is Boltzmann constant we assumed that the final temperature of the T-jump was the operational temperature for the kinetics Besides the lesser configurational flexibility of H-DNA replacement of H with water molecules also contributes to the entropic change resulting in a ${\Delta}S_{\mathrm{d}}^\dagger$ value for native DNA that is half of value for H-DNA b Distribution of the H1–N3 distance in the GC termini shown in (c) The distribution with H1–N3 larger than 3 Å (marked with pink dashed lines) are multiplied by a scaling factor of 10 d The H1–N3 distance in the GC termini at 60 and 70 °C as a function of the trajectory length e Configuration of the terminal G:C pair when the N1–H3 distance deviates to 15 Å at 60 °C (e f Average minor-groove widths for the DNA structure throughout the trajectory at 27 Nucleobases involved in forming hydrogen bonds with H are marked with orange boxes A water molecule plays a role in separating the G:C termini through hydrogen bonding and gives rise to the plateau at ~5.5 Å in the range of 1–1.6 µs at 70 °C finding that the ligand’s impact on fraying is correlated with its binding affinity they observed that H binding suppressed the short time dissociation response (~100 ns) from A ring modes they concluded that end-fraying is suppressed by ligand binding based on the observation that the ~100 ns fraying of the G:C termini showed a larger change in DNA without H bound than for H-DNA We believe these conclusions are influenced by the observation window for the experiment when the full dissociation time-scales we observe are more than an order of magnitude slower These observations suggest that water molecules interrupt the hydrophobic region in H-DNA by breaking the hydrogen bonds of H with the T carbonyls The rate distribution of this binding signal and the A:T dissociation response highlights the significance of the hydrophobic interactions in stabilizing the complex although the electrostatic attraction between the charged ligand and DNA backbone is also an important stabilizer of the minor-groove structure in the T8–T9 tract we have demonstrated that the small ligands binding can strongly disturb the stability of base pairs adjacent to the binding site with the observation that the percentage of dissociated G:C pairs is 66% in H-DNA instead of 17% in native DNA within the first 100 ns after T-jump given the strong stabilization effect of ligand on dsDNA which increases the melting temperature by 45 °C and dramatically slows down the dissociation of dsDNA with the observed time being 50 times larger than that of native DNA The hydrogen bonding is found to be essential in stabilizing H-DNA by seeing that the hydrogen bonds between H and thymines break prior to the dissociation of central A:T pairs Our thermodynamic analysis suggests that H works concertedly with ssDNA in the formation of H-DNA Under conditions that dsDNA is slightly unstable (the fraction of ssDNA >1%) the H+dsDNA association description shows obvious failure in predicting the fraction of H-DNA indicating that the association of H-DNA is a concerted process The association/dissociation free energy change is more appropriate than the binding constant for characterizing the binding affinity between ligands and the DNA binding site The experimental and analytical approach developed here can further help in elucidating the binding mechanism of other DNA binders and provide a different perspective in the design of new type derivatives for extensive applications in biotechnology The DNA oligonucleotide used in this study is the self-complementary sequence 5′-CGC AAA TTT GCG-3′ purchased from Integrated DNA Technologies (IDT) at desalt-grade purity and further purified by dialysis using membrane tubing with a 0.5 kD MWCO (Spectrum Micro Float-A-Lyzer) and distilled water at 0 °C for 8 h Hoechst 33258 (H) was purchased from Chemodex and is used as received The duplex DNA bound with Hoechst 33258 is denoted by H-DNA all DNA samples were prepared in pH 7.4 deuterated buffer with 5 mM sodium phosphate and 200 mM sodium chloride pH values were adjusted with 1 M HCl and NaOH solutions and then checked with pH meter (Fisherbrand accumet AB150) The concentration of oligonucleotide and H was confirmed on a NanoDrop UV/vis spectrometer (Thermo Scientific) H-DNA complexes were prepared by mixing buffer solutions in a 1:1 dsDNA-to-H mole ratio We use CD to refer to the total concentration of native and H-DNA (the equivalent value of dsDNA) all samples were annealed by heating to 95 °C for 3 min and then cooling gradually to room temperature over 10–15 min labile protons of DNA and H were HD exchanged in deuterated water (D2O Cambridge Isotopes) and lyophilized before dissolving into the deuterated buffer Fluorescence experiments were carried out using a Horiba Fluorolog-3 at 1 nm resolution The temperature-dependent fluorescence was recorded at 446 nm The 0.2 and 2 µM samples were prepared via serial dilution from the original 1 mM solution A quartz cuvette with 0.5 cm path length is used for all fluorescence measurements samples were held between two CaF2 windows with a 50 μm Teflon spacer defining the path length and mounted in a home-built brass sample cell connected to a recirculating chiller to control the sample temperature the bath temperature was ramped between 0 and 105 °C in 3 °C steps and the temperature of the sample was recorded with a thermocouple embedded in the brass sample jacket Spectra were recorded on a Bruker Tensor 27 FTIR spectrometer at 4 cm–1 resolution averaging 30 scans at each temperature step after waiting 75 s for the sample cell to equilibrate at each bath set point experiments are performed in the boxcar geometry with three variably time-delayed pulses generating the nonlinear signal and a fourth reference pulse used for balanced heterodyne spectral interferometry The nonlinear signal was collected at a fixed waiting time τ2 = 150 fs as a function of evolution time τ1 scanned in 4 fs steps out to 2500 and 2000 fs for rephrasing and non-rephasing spectra The transient heterodyned dispersed vibrational echo (t-HDVE) and transient 2D-IR (t-2D-IR) data were collected over a time window from 5 ns to 50 ms after a 15 °C T-jump which is limited by the thermal re-equilibration time of solvent we coated the CaF2 windows with 12.5 µm Dupont FEP fluorocarbon film using vacuum oven (Fisherbrand Isotemp Model 281A) An optical quality coating was achieved by heating the film attached windows at 290 °C for 30 min under vacuum The t-HDVE spectra were collected using the Fourier transform spectral interferometry method with the local oscillator stepped from −10 to 10 fs in 5 fs steps τ1 was undersampled in 16 fs steps from −60 fs to 1750 and 1250 fs for rephasing and non-rephasing surfaces t-HDVE time traces and t-2D-IR surfaces represent at least five averaged data sets The polarization was set to ZZZZ for all nonlinear IR measurements All anlaysis and simulations were performed using available softwares and packages mentioned in the “Methods” sample size 1 mL was used and each data point is the average of five replicates The number of replicates is 32 for the equilibrium IR spectra and 8 for the transient IR spectra All the above mentioned experimental dataset were repeated at least once to confirm their reproducibility Further information on research design is available in the Nature Research Reporting Summary linked to this article All data supporting the findings of this study are available within the Article and its Supplementary Information and/or from the corresponding author upon reasonable request. Source data underlying plots shown in figures are provided in Supplementary Data 1 The mutagenic properties of DNA minor-groove binding ligands Regulation of gene expression by small molecules Using genome sequence to enable the design of medicines and chemical probes Binding characteristics of Hoechst-33258 with calf thymus DNA and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight-binding with a wide spectrum of site affinities Massive parallel analysis of DNA-Hoechst 33258 binding specificity with a generic oligodeoxyribonucleotide microchip Rapid screening of DNA-ligand complexes via 2D-IR spectroscopy and ANOVA-PCA Water at DNA surfaces: ultrafast dynamics in minor groove recognition Detection of DNA-ligand binding oscillations by Stokes-shift measurements The role of water H-bond imbalances in B-DNA substate transitions and peptide recognition revealed by time-resolved FTIR spectroscopy Water-mediated binding of agents that target the DNA minor groove Origin of DNA-induced circular dichroism in a minor-groove binder Specific binding of hoechst 33258 to the d(CGCAAATTTGCG)2 duplex: calorimetric and spectroscopic studies Binding of a desmetallo-porphyrin conjugate of Hoechst 33258 to DNA Strong binding to single-strand oligonucleotides DNA-Sequence Preferences of Several at-Selective Minor-Groove Binding Ligands Ensemble and single-molecule fluorescence spectroscopic study of the binding modes of the bis-benzimidazole derivative Hoechst 33258 with DNA DNA structural features responsible for sequence-dependent binding geometries of Hoechst 33258 The different binding modes of Hoechst-33258 to DNA studied by electric linear dichroism Multiple binding modes for dicationic Hoechst 33258 to DNA Thermodynamics of DNA minor groove binders Three-dimensional crystal structure of the A-tract DNA dodecamer d(CGCAAATTTGCG) complexed with the minor-groove-binding drug Hoechst 33258 Dynamical disorder in the DNA hydration shell Ultrafast vibrational dynamics and local interactions of hydrated DNA Association of the minor groove binding drug Hoechst 33258 with d(CGCGAATTCGCG)(2): Volumetric Hydration changes in the association of Hoechst 33258 with DNA Atomistic account of structural and dynamical changes induced by small binders in the double helix of a short DNA 2D-IR spectroscopy shows that optimized DNA minor groove binding of Hoechst33258 follows an induced fit model Recognition of the four Watson-Crick base pairs in the DNA minor groove by synthetic ligands Kinetics of binding of Hoechst dyes to DNA studied by stopped-flow fluorescence techniques DNA minor-groove recognition by small molecules Force measurements reveal how small binders perturb the dissociation mechanisms of DNA duplex sequences Monitoring base-specific dynamics during melting of DNA-ligand complexes using temperature-jump time-resolved infrared spectroscopy Anharmonic vibrational modes of nucleic acid bases revealed by 2D IR spectroscopy Sequence-dependent effects in drug-DNA interaction: the crystal structure of Hoechst 33258 bound to the d(CGCAAATTTGCG)2 duplex Protonation equilibria of Hoechst 33258 in aqueous solution Real-time atomistic description of DNA unfolding Induced fit DNA recognition by a minor groove binding analogue of Hoechst 33258: fluctuations in DNA A tract structure investigated by NMR and molecular dynamics simulations Sequence-Dependent Mechanism of DNA Oligonucleotide Dehybridization Resolved through Infrared Spectroscopy On the rate distribution analysis of kinetic data using the maximum entropy method: Applications to myoglobin relaxation on the nanosecond and femtosecond timescales Direct observation of activated kinetics and downhill dynamics in DNA dehybridization The molecular-structure of the complex of Hoechst-33258 and the DNA dodecamer D(Cgcgaattcgcg) Isohelicity and phasing in drug–DNA sequence recognition: crystal structure of a tris(benzimidazole)–oligonucleotide complex Electrochemical studies for the interaction of DNA with an irreversible redox compound - Hoechst 33258 Comparison of the electrophoretic and hydrodynamic properties of DNA and RNA oligonucleotide duplexes Diffusion coefficient of DNA molecules during free solution electrophoresis Parmbsc1: a refined force field for DNA simulations The missing term in effective pair potentials The dynamics of water at DNA interfaces: computational studies of Hoechst 33258 bound to DNA PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data Conformational analysis of nucleic acids revisited: Curves Download references We thank Paul Sanstead and Brennan Ashwood for helpful discussion and experimental assistance with T-jump experiments and Elena Solomaha of the University of Chicago BioPhysics Core Facility for measurements on the Horiba Fluorolog-3 spectrometer thank the National Institute of General Medical Sciences of the National Institutes of Health (Award No are grateful for computing resources at the Center for Research Computing at the University of Notre Dame designed and performed all the experiments All authors discussed the results and commented on the manuscript Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s42003-020-01241-4 Metrics details Cell-permeable DNA stains are popular markers in live-cell imaging Currently used DNA stains for live-cell imaging are either toxic require illumination with blue light or are not compatible with super-resolution microscopy is applicable in different cell types and tissues and is compatible with super-resolution microscopy The combination of these properties makes this probe a powerful tool for live-cell imaging Building on these findings we introduce here a bisbenzimide–SiR conjugate and demonstrate its utility as DNA stain for live-cell (super-resolution) microscopy (b) Titration of 0.1 μM SiR–Hoechst probe with varying concentrations of hairpin DNA or hairpin RNA of three independent replicates measured in triplicates (c) Excitation spectra and absorbance spectra of 1 μM SiR–Hoechst in presence of 50 μM hairpin DNA or RNA Determined excitation maximum at 652 nm and emission maximum at 672 nm (a) Long-term live-cell microscopy with SiR–Hoechst and other far-red DNA dyes HeLa cells were imaged for 24 h on a wide-field epifluorescence microscope in presence of the dyes at the indicated concentrations The contrast was linearly adjusted for the different dyes as indicated relative to the reference contrast used to display SiR–Hoechst (0.5 μM) (b) Quantification of nuclear-staining intensity and specificity Nuclei were automatically segmented based on the H2B-mCherry channel and the mean fluorescence intensity was then quantified in the channel used for imaging far-red DNA dyes The staining specificity was calculated as the ratio of nuclear mean fluorescence divided by cytoplasmic mean fluorescence quantified in a narrow area surrounding the nucleus Spots indicate mean and bars indicate s.e.m of three independent biological replicates; >1,108 cells per replicate (c) Quantification of cell proliferation based on the last and the first frames of the live-cell movies shown in a To assess phototoxicity caused by time-lapse imaging separate wells in the imaging plate were exposed to fluorescence excitation light only at 0 and 24 h time points of three independent biological replicates; >76 cells per replicate To quantify cells with no or low staining in the far-red channel a stably expressed histone 2B (H2B)-mCherry was used as a reference chromatin marker (d) Images of different cell types with 4 μM SiR–Hoechst SiR–Hoechst provides a bright and highly specific nuclear stain in live cells SiR–Hoechst did not impair cell proliferation within the 24-h measurement interval, up to the highest concentration tested (25 μM; Fig. 2c). DRAQ5, Ruby and SYTO 61, in contrast, were highly toxic at 500 nM (Fig. 2c) Without regular fluorescence excitation light exposure by time-lapse microscopy the toxicity was reduced for DRAQ5 and Ruby This indicates that these three dyes substantially sensitize cells for phototoxicity which was imaged under identical conditions DRAQ5 and Ruby strongly inhibited cell proliferation even in the absence of repetitive exposure to light by time-lapse microscopy SiR–Hoechst outperforms the commercially available far-red DNA stains SYTO 61 Vybrant DyeCycle Ruby and DRAQ5 both in terms of labelling specificity as well as minimal toxicity (a) HeLa cells stained with 200 nM SiR–Hoechst were imaged over 3.4 h with a time lapse of 4.8 min Images show maximum intensity projections of five Z-sections To quantify control cells without staining in the far-red channel (b) Mitotic duration was measured as the time from prometaphase onset until anaphase onset based on visual inspection of the image data (c) Chromosome missegregation was quantified as the number of anaphase events with lagging or bridged chromosomes divided by the total number of anaphase events SiR–Hoechst was present in culture media throughout the entire imaging duration (a) Confocal and (b) STED images of living HeLa cell nuclei stained with 4 μM SiR-Hoechst for 2 h in phenol red-free medium Both confocal and STED images were slightly smoothed A part of the background in the STED image is caused by a weak but non-negligible two-photon excitation of Hoechst by the 775 nm STED beam close-ups of the regions indicated with a box (c) Examples of normalized fluorescence intensity profiles obtained in the regions indicated by the arrows in b Profiles from the raw STED images were fitted to Gaussian distributions Number corresponds to single measurement of full width at half maximum of the fitted peak Here we demonstrate that DNA-binding ligands such as the bisbenzimides can also be employed these data make a strong case for carboxylated SiR as first choice when coupling ligands to fluorophores for the generation of fluorescent probes for live-cell imaging excellent biocompatibility and applicability to STED microscopy make SiR–Hoechst a powerful tool for the imaging of DNA in living cells and tissues we purchased analogous hairpin RNA for binding studies Synthetic oligonucleotides were dissolved in Tris-buffered saline (TBS; 50 mM Tris-HCl Hairpin formed by putting the tube with DNA or RNA solution into boiling water bath which was slowly cooled down to room temperature KD measurements were performed by titrating the SiR–Hoechst (100 nM in 150 mM TBS with 1 mg ml−1 BSA) with increasing concentrations of the 28-bp hairpin DNA or its RNA analogue in a 96-well plate and measuring the increase in fluorescence (λex=640 and λem=670 nm) on a plate reader after 2–3 h incubation at room temperature The KD values were determined by plotting the emission intensity versus the DNA or RNA concentration and fitting the curve in Graphpad Prism 6 to the ‘one site—specific binding’ function each time technical triplicates were measured All the used cell lines were cultured in DMEM (Life Technologies 61965-059) supplemented with 10% foetal calf serum (FCS) (FCS A15-151) and pen/strep (100 units per ml and 100 μg ml−1 cells were stained with the probes at 37 °C in HDMEM (phenol red-free DMEM—Invitrogen 31053-028—buffered with 10 mM HEPES) supplemented with 10% FCS and pen/strep Imaging was performed in HDMEM buffer with 10% FCS For regular microscopy cells were stained in DMEM growth medium at 37 °C supplemented with 10% FCS A HeLa (‘Kyoto’ strain) cell line stably co-expressing H2B-mRFP and MyrPalm-mEGFP18 was used for live-cell microscopy experiments H2B-mRFP was imaged as a reference marker to quantify cell proliferation mitotic duration and chromosome missegregation in the control cells (dimethylsulfoxide) that were not stained with SiR–Hoechst HeLa cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) 0.5 μg ml−1 puromycin and 500 μg ml−1 G418 cells were grown either in LabTek II chambered coverslips (ThermoScientific) or 96-well plastic-bottom plates (μclear; Greiner Bio-One Ltd.) in DMEM containing 10% (v/v) FBS and 1% (v/v) penicillin–streptomycin but without riboflavin and phenol red to reduce background fluorescence For wide-field and confocal time-lapse imaging SYTO 61 (Life Technologies) and Vybrant DyeCycle Ruby (Life Technologies) were added between 30 min and 2 h before imaging at the concentrations as indicated in the main text and imaged in Clone 8 medium enriched with 1.6 μM SiR–Hoechst Imaging of stained cells was performed on Leica DMI6000B microscope equipped with a Leica HCX PL APO × 100 1.47 oil objective and a Hamamatsu-C9100 EM-charge-coupled device camera (512 × 512 pixels) (Fig. 2d) The following dichroic mirror and filters were used for SiR signal detection: excitation BP 635/30 emission BP 700/72 and dichroic mirror at 650 nm Z-stacks with voxel size of 240 × 240 × 692 nm were acquired and images were presented as maximal intensity projections using a 10 × 0.5 numerical aperture (NA) S Fluor dry objective (Nikon) and reflection-based laser autofocus The following dichroic mirror and filters were used for SiR signal detection: excitation BP 640/30 emission BP 690/50 and dichroic mirror at 660 nm A microscope stage incubator was used to maintain cells in humidified atmosphere of 5% CO2 at 37 °C For laser scanning confocal imaging (Figs 2a and 3a and Supplementary Movie 1) a customized Zeiss LSM780 microscope equipped with a × 40 1.4 NA oil DIC Plan-Apochromat objective (Zeiss) was used controlled by ZEN 2011 software and an autofocus macro (AutofocusScreen) kindly provided by J Ellenberg (European Molecular Biology Laboratory The following laser illumination and fluorescence emission detection was used for SiR imaging: 633 nm laser excitation emission detected on 32-channel gallium arsenide phosphide (GaAsP) detector array at wavelengths between 647 and 691 nm a humidified atmosphere of 5% CO2 and 37 °C was provided by a European Molecular Biology Laboratory incubation chamber Imaging of Drosophila notum epithelium (Supplementary Fig. 2 and Supplementary Movie 2) was performed using a 3i Marianas spinning disk confocal set-up based on a Zeiss Z1 stand a × 63 PLAN APO NA 1.4 objective and a Yokogawa X1 spinning disk head followed by a × 1.2 magnification lens and an Evolve EMCCD camera (Photometrics) Jupiter-GFP was detected using a 50-mW 488-nm excitation laser and a 520/30 bandpass emission filter SiR–Hoechst was detected using a 50-mW 633-nm excitation laser and a 647-nm long pass filter Fast Z-stack acquisition of entire notum (66.5 μm by 0.5 μm increment) was obtained using a piezo stage (Mad City Labs and the resulting stack was projected using maximum intensity projection The images shown in Fig. 4 were taken on an Abberior Instruments QuadSCAN STED microscope a 640 nm excitation line and avalanche photodiode (APD) gated detection The images shown in Supplementary Fig. 3 were taken on Leica SP8 gSTED × 3 microscope equipped with HC PL APO CS2 × 100/1.40 oil objective and 775 nm STED laser Excitation white light laser was set to 633 nm and signal was detected by HyD detector set to 650–700 nm interval with 0.3–6.0 ns time gating images were acquired with three times line averaging and pixel size in xy plane was 20 × 20 nm mitotic duration and chromosome missegregation were scored by visual inspection of time-lapse movies Proliferation was calculated as the ratio of all live cells in the last movie frame (24 h) divided by all live cells from the first movie frame Mitotic duration was defined as the interval from prometaphase onset until anaphase onset for all cells entering mitosis within the first 12 h of the experiment and determined based on the chromatin morphology The incidence of chromosome missegregation was calculated by dividing the number of anaphase cells with lagging or bridged anaphase chromosomes by the total number of anaphase cells Cell nuclei were automatically segmented in five consecutive image frames per experimental condition by local adaptive thresholding in the H2B-mCherry channel Mitotic cells and dead cells were excluded from analysis based on automated classification by supervised machine learning The mean fluorescence was then measured in the far-red channel To calculate the nucleo/cytoplasmic fluorescence ratio a cytoplasmic region was defined for each cell as a 5 pixel wide rim around the nuclear segmentation mask Extracellular background fluorescence was manually measured and subtracted from intracellular mean fluorescence measurements The nucleo/cytoplasmic fluorescence ratio was first calculated for individual cells to derive the mean nucleo/cytoplasmic fluorescence ratio of the cell population was then calculated for each dye condition based on three independent biological replicates Fluorescence cross-talk from the H2B-mCherry channel was very low (<2% of the signal detected in 500 nM SiR–Hoechst-treated cells) as assessed by measuring fluorescence intensity in dimethylsulfoxide-treated control cells SiR–Hoechst is a far-red DNA stain for live-cell nanoscopy Automated microscopy for high-content RNAi screening Phototoxicity of Hoechst 33342 in time-lapse fluorescence microscopy Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe in intact human cells analyzed by flow cytometry Fluorogenic probes for live-cell imaging of the cytoskeleton A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins Hoechst tagging: a modular strategy to design synthetic fluorescent probes for live-cell nucleus imaging migration and protein kinase C activity in human retinal pigment epithelial cells Antiproliferative effect of verapamil alone on brain tumor cells in vitro The fluorescent toolbox for assessing protein location and function Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo Optimized near-IR fluorescent agents for in vivo imaging of Btk expression A general method to improve fluorophores for live-cell and single-molecule microscopy STED nanoscopy reveals the ubiquity of subcortical cytoskeleton periodicity in living neurons A general approach to spirolactonized Si-rhodamines Red Si-rhodamine drug conjugates enable imaging in GFP cells Super-resolution imaging of the Golgi in live cells with a bioorthogonal ceramide probe Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex Aurora B-mediated abscission checkpoint protects against tetraploidization Monitoring notch/delta endosomal trafficking and signaling in Drosophila CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging Download references We thank Jan Keller (MPI Göttingen) for participating in the analysis of the super-resolution images and Rudolf Höfler (IMBA Vienna) for help with the analysis of live-cell microscopy data The Johnsson Laboratory acknowledges support from the Swiss National Science Foundation and the NCCR Chemical Biology The Gerlich laboratory has received financial support from the European Community’s Seventh Framework Programme FP7/2007-2013 under grant agreement no from an ERC Starting Grant (agreement no 281198) Forschungs- und Technologiefonds (WWTF; project no and from the Austrian Science Fund (FWF; project no Ecole Polytechnique Fédérale de Lausanne (EPFL) Institute of Chemical Sciences and Engineering (ISIC) Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA) Claudia Blaukopf & Daniel Wolfram Gerlich Emmanuel Derivery & Marcos Gonzalez-Gaitan Max-Planck-Institute for Biophysical Chemistry did the experiments; all authors contributed to manuscript writing and data analysis have filed a patent application on SiR derivatives The remaining authors declare no competing financial interests Supplementary Methods and Supplementary References (PDF 443 kb) High resolution time-lapse confocal imaging of Hela cells Cells were stained with 200 nM SiR-Hoechst before imaging SiR-Hoechst imaging in the Drosophila Notum Time-lapse spinning disk confocal imaging of Drosophila notum epithelium showing Jupiter-GFP Maximum intensity projection of 133 planes with an increment of 0.5 μm (66.5 μm total) This movie corresponds to Supplementary Figure 1 Download citation KARACHI: Standard Chartered Bank Pakistan has been awarded Hoechst Pakistan’s cash management mandate through the signing of a memorandum of understanding (MoU) at the bank’s head office This agreement positions SCB as the exclusive collections bank for Hoechst Pakistan enabling digital transactions 24/7 with nearly 30 banks The partnership allows Hoechst Pakistan to streamline its cash management processes enhancing operational and cost efficiencies Hoechst will now be able to match receipts with invoices reconcile collections across multiple product lines and manage its financial operations with greater ease and control -- without the need for multiple physical accounts The MoU was signed by Head of Client Coverage and Commercial Banking (CIB) Pakistan at Standard Chartered Arslan Nayeem and Chief Executive Officer of Hoechst Pakistan Sajjad Iftikhar The event was attended by senior representatives from both organizations including Regional Head of Transaction Banking Sales (MEPA) at Standard Chartered Motasim Iqbal and Chief Financial Officer of Hoechst Pakistan Yasser Pir Muhammad “We are delighted to take on this important cash management mandate in the pharmaceutical sector has announced its decision to establish a wholly owned subsidiary in the United Arab Emirates (UAE) marking another step by Pakistani firms towards making the most of the Gulf state’s business-friendly environment In a notice to the Pakistan Stock Exchange (PSX) on Friday the pharmaceutical company disclosed that its board of directors approved the move during a meeting held on December 19 “The subsidiary will be primarily engaged in commercial trading with import and warehousing as its ancillary activities,” the company stated adding that the incorporation is subject to all necessary regulatory approvals The UAE has increasingly become a preferred destination for Pakistani businesses due to its streamlined payment processes The Gulf state ranks ninth globally in enforcing contracts out of 190 countries and holds the top spot for ‘getting electricity,’ according to international metrics several Pakistani companies have announced their expansion into the UAE revealed plans to establish a subsidiary in the region Treet Corporation Limited successfully incorporated Treet Trading LLC in Dubai Industry experts view such moves as strategic enabling Pakistani companies to tap into the UAE’s robust infrastructure The Gulf state’s position as a regional hub offers opportunities for businesses to scale operations and reach international markets efficiently Hoechst Pakistan’s expansion into the UAE underscores the growing trend of Pakistani firms seeking to capitalise on the advantages offered by one of the Middle East’s most dynamic economies A service livestream will be available at the time of the service at this link https://www.youtube.com/watch?v=1CBXYeBNKKM the fourth of four children to Verna and Waldo He graduated from Bismarck High School in 1974 and from the North Dakota School of Science in 1976 with an associate degree in Diesel Mechanics was born followed by Lucas in 1977 and Brandon in 1979 In 1982 he married Wanda Baar and in 1994 Rose was born Vern was an exceptionally hard worker and was very proud of the life he built with Wanda In his early career he worked for Burlington Northern until they moved to Bismarck where he worked for Bobcat especially after Wanda passed away in 2019. He was a dedicated member of the Freedom Riders Motorcycle Club for over 40 years He loved riding with his club brothers as well as his two sons He was well respected in the Freedom Riders Club as well as in the motorcycle community across the state His collections included larger items like cars and motorcycles as well as smaller collectibles such as Budweiser memorabilia and cans Vern was grateful to have shared his life with his two daughters Nikki (Jason) Van Lishout and Rose (Nolan) Schwarz; his sons Lonnie and Mark (Marlya) Hoechst; many nieces and nephews; and his beloved cat Metrics details Cell quantification is widely used in basic or applied research The current sensitive methods of cell quantification are exclusively based on the analysis of non-fixed cells and do not allow the simultaneous detection of various cellular components sensitive and cheap method of the quantification of fixed adherent cells is described here It is based on the incubation of DAPI- or Hoechst 33342-stained cells in a solution containing SDS The presence of SDS results in the quick de-staining of DNA and simultaneously in an up-to-1,000-fold increase of the fluorescence intensity of the used dyes This increase can be attributed to the micelle formation of SDS The method is sufficiently sensitive to reveal around 50–70 human diploid cells It is compatible with immunocytochemical detections the detection of DNA replication and cell cycle analysis by image cytometry The procedure was successfully tested for the analysis of cytotoxicity The method is suitable for the quantification of cells exhibiting low metabolic activity including senescent cells The developed procedure provides high linearity and the signal is high for at least 20 days at room temperature Only around 90 to 120 minutes is required for the procedure’s completion Cell quantification is a common task for many laboratories A typical example of its use is drug-discovery research They are usually based on the time-consuming direct calculation of cells using e.g a haemocytometer or a much easier determination of the relative cell concentrations Since the determination of the relative concentrations of cells is sufficient in many studies these methods are a common tool for routine cell quantification the absolute number of cells can be determined after the calibration of the signal using samples containing a known number of cells it commonly results in the non-linear dependence of the signal on the cell number The dependence on the metabolic state can also result in low sensitivity if the metabolically less active cells the specific conditions have to be found for the individual cell lines The methods based on DNA staining do not depend on the cell metabolism and some of them exhibit sufficient sensitivity to reveal several tens of cells cell lysis is usually required for maximal sensitivity and the linearity of the dependence of the signal on the cell number It can result in the substantial prolongation of the procedure and/or additional costs the stability of the signal is usually low and requires relatively fast evaluation of the processed cells or freezing of the sample we have developed an approach for the quantification of the fixed cells that does not require cell lysis as the DNA dye is eluted from cellular DNA using an elution solution It results in a highly homogeneous solution of the eluted DNA dye and substantial increase of its fluorescence intensity only a fraction of the sample can be measured without any decrease in the accuracy of the measurement the cell cycle analysis by image cytometry or the detection of various cellular components can be performed before the elution step It provides the possibility to quantify the cell number in various vessels including well plates Petri dishes or even coverslips at reasonable cost The scheme of the procedure with the recommended incubation times for Hoechst staining is shown the incubation in step 4 (the washing step) should be shortened to 3 × 2 minutes The other incubation times are the same as in the case of Hoechst dyes but it is highly recommended if 96-well plates are used We compared the signal in samples fixed with 70% ethanol or 100% methanol at various temperatures (−20 °C 0 °C or RT) and for various times (10 minutes fixation by air-drying and formaldehyde fixation With the exception of formaldehyde fixation the rest of the fixation protocols used exhibited similar signals The 10-minute fixation with 2% formaldehyde provided around half of the signal obtained by the ethanol or methanol fixation or by air-drying As the 10-minute fixation with 70% ethanol at RT resulted in the effective protection of the cells from loosening from the cultivation surfaces and ethanol is less toxic than methanol we used ethanol fixation in all the subsequent experiments the ethanol fixation enables interruption of the procedure for a relatively long time the storage of samples for 3 months in 70% ethanol at −20 °C was successfully tested without any impact on the signal intensity the procedure can be interrupted for several months after ethanol fixation Drying of the cells is performed immediately after ethanol fixation it is highly recommended if the cells are incubated in 96-well plates It allows stronger attachment of the cells to the cultivation surface and protects the cells from loosening during the washing steps as the repeated fluid stream from the pipette tip can release a substantial portion of the cells from the well The cell drying after ethanol aspiration typically lasts 20 to 30 minutes depending on the temperature humidity and the amount of ethanol volume resting in the well It can be easily controlled as ethanol remnants are visible at the bottom of the well plates This step is typically useless if plates with larger wells or Petri dishes or culture flasks are used The cell drying should not exceed one hour progressively as extensive drying can result in the necessity of prolonging the elution step 200 µl of the 2–3 µM dye solution is sufficient for staining cells grown on 1 cm2 of the cultivation area we used 100 µl of 3 µM DAPI or 2 µM Hoechst dye per well If a Petri dish with the diameter of 3.5 cm was used The additional increase of the dye concentration can result in the non-specific binding of the dye to the surface of the culture vessels and also to the non-linear dependence of the signal on the cell number the lower concentrations can result in a lower signal per cell if a high density of cells is reached due to the depletion of a substantial portion of the dye from the solution Thirty minutes is the optimal time with respect to the speed and the signal intensity longer incubation times can be used without any effect on the results the shorter times can result in a lower signal and lower sensitivity as the shaking accelerates the binding of the dye to the DNA The impact of RNase A on the signal of Hoechst 33258 or Hoechst 33342 or DAPI is shown dried and incubated in 1× PBS with or without RNase A for 1 hour at 37 °C and the developed approach was used The signal is normalised to the signal measured in samples without RNase A treatment which is equal to 100% the non-specifically bound dyes are removed Dyes can be non-specifically bound to the cells or to the surfaces of the culture vessels the surfaces of the common polystyrene cultivation vessels bind all tested DNA dyes These non-specifically bound dyes have to be removed before the elution step otherwise it will result in a lowering of the sensitivity of the approach As a 40-minute washing time is usually necessary to remove a substantial portion of the DNA dyes from the culture surfaces using 1× PBS we tested other solutions to accelerate this step we found that the use of solutions with a low pH and relatively high ionic strength results in the very fast dissociation of the dyes from the cultivation surfaces the solution of 50 mM citric acid supplemented with 200 mM CaCl2 and 0.2% Tween 20 makes it possible to wash out nearly all the surface-bound Hoechst molecules in several minutes three washing steps each for five minutes are sufficient for the removal of the majority of the non-specific signal the prolongation of the washing time did not result in the lowering of the DNA-specific signal of Hoechst dyes and this step can be prolonged according to the user needs Additional tests have shown that the linearity of the signal dependence on the cell number can be significantly improved by the addition of 2 M NaCl to the washing solution It is probably a result of the washing out of the non-specifically bound dye from the cellular components we successfully tested a solution of 20 mM citrate buffer pH 5 supplemented with 0.5 M NaCl and 0.2% Tween 20 As DAPI is partially removed also from cellular DNA in the case of low pH and the high ionic strength the total time of the washing step should not exceed 15 minutes it can result in the progressive lowering of the signal the washing solution contained 2 mM CuSO4 as well The addition of copper sulphate efficiently protects the ethanol- methanol-fixed cells and also air-dried cells from SDS-induced lysis The cell lysis is accompanied by the progressive increase of the solution viscosity and difficulties with the transfer of the solution of the eluted dyes to the black well plates No such treatment is necessary if formaldehyde-fixed cells are used Although the incubation with copper ions can be performed at any time before the elution step the addition of copper ions into the washing solution is advantageous as it does not result in the prolongation of the procedure The dependence of the signal on the SDS and dye concentrations (a) The dependence of the signal of the 1 µM solution of DAPI or Hoechst dyes on the percentage concentration of SDS in various buffers is shown The signal is normalised to the signal measured in the samples with the DNA dye diluted in 2% SDS and a phosphate buffer (b) The dependence of the signal enhancement of DNA dyes on the SDS concentration in various buffers is shown The values of enhancement are expressed as a multiple of the signal intensity of the sample without the addition of SDS (c) The dependence of the signal on the concentration of DNA dyes in 2% SDS is shown The log scales of both dye concentrations and signal intensity are used (d) The dependence of the signal of 1-µM solution of DAPI or Hoechst dyes on the percentage concentration of SDS in water is shown The signal is normalised to the signal measured in samples with DNA dye diluted in 0.25% SDS in water which is equal to 100% buffered solution of anionic detergent - SDS This step assures the elution of the DNA dyes bound to the DNA in the solution necessary to determine at which concentration of SDS the signal will be the highest whether it will be sufficient for the precise determination of the relative cell number whether there is a cross-section between the concentration which is necessary for the elution and the concentration for the sufficient increase of the signal and how linear the dependence of the signal is on the cell number in a sample the maximal fluorescent signal overlaps with values which are necessary for the elution of dyes from the DNA of the samples and at the same time the fluorescent signal linearly increases with the increasing concentration of the dye and cells the minimal concentration leading to the elution of the DNA dyes this concentration required more than one-hour incubation for the effective elution of the dye from the DNA a solution of 2% SDS resulted in the effective elution of the DNA dyes in less than 15 minutes The course of the signal dependence on SDS concentration in the case of Hoechst dyes in phosphate buffer pH = 7 and DAPI in all the tested buffers showed that the signal intensity is similar in a wide range of SDS concentrations including the concentration suitable for the elution of both Hoechst dyes or DAPI from DNA The same experiment showed that the addition of SDS resulted up to 1,000-fold increase of the fluorescence intensity in the case of Hoechst 33342 and more than 700-fold increase in the case of Hoechst 33258 (Fig. 3b) Only an approximately 90-fold increase of the signal was measured if DAPI was used The dependence of the signal of both Hoechst dyes and DAPI in the elution solution containing 2% SDS on the dye concentration was linear in all the tested concentrations (Fig. 3c) Agitation of the samples at 300 rpm and RT is sufficient for the elution of DNA dyes from DNA in 15 minutes 120 µl per well of a 96-well plate and 0.5 ml per Petri dish with a diameter of 3.5 cm was successfully tested 100 µl of the elution buffer containing the eluted DNA dye was carefully transferred to the black 96-well plates to maximize the signal/noise ratio and the signal was measured using the plate reader The formation of bubbles should be avoided during the transfer as it can result in false results The signal can be measured immediately after the transfer of the solution to the black well plate or, if necessary, the black well plate can be sealed by parafilm and stored at RT until the measurement is performed. In this respect, the tested samples provided high fluorescent signal for at least twenty days (Supplementary Fig. S2) Dependence of the signal on the cell number (a) The microscopic images of the HeLa cells grown in the concentration of ca 71,000 cells per well of the 96-well plate Cell nuclei labelled with DAPI (left) and the phase contrast image of the same cells (right) is shown (b) The dependence of the signal provided by the developed approach using DAPI or Hoechst 33342 and by MTT assay on the number of HeLa cells is shown The highest HeLa concentration was 71,000 cells/well of the 96-well plate The signal is normalised to the signal measured in samples with the highest cell number which is equal to 100% The black dotted line intersects the curves at the point corresponding to 50% of the maximal signal (c) The dependence of the signal provided by the developed approach using DAPI or Hoechst 33342 and by MTT assay on the number of HeLa cells is shown The highest HeLa concentration was 35,500 cells/well of the 96-well plate We compared the linearity of the approach using DAPI or Hoechst 33342 staining with the approach based on the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). From the graph of HeLa cells (Fig. 4b) it is evident that the highest linearity was provided by DAPI staining (linear regression then by Hoechst 33342 staining (second-order polynomial regression R2 > 0.9953) and the lowest by MTT (one-phase association regression half of the maximal signal corresponded to 50% 30% or 15% of the number of cells in the case of DAPI If the maximal cell confluence decreased to 50% (35,500 cells per well), the highest linearity was provided by Hoechst 33342 dye (Fig. 4c, linear regression, R2 > 0.9994. DAPI and especially MTT provided lower linearity (Fig. 4c) this dye is much less appropriate for cell quantification than Hoechst 33342 We also tested re-staining the cells after the elution of the DNA dyes from the DNA this protocol provides similar data as was provided by the first staining and elution it requires at least four washing steps with 20 mM Tris-HCl The total length of this washing should be at least 30 minutes at least a 2-hour incubation with DNA dyes is necessary This time should even be prolonged to 4 hours if highly confluent cell populations are used the cells with the higher confluence will contain a lower amount of the dye per cell than cells with lower confluence (a) The sensitivity of the developed approach using either DAPI or Hoechst 33342 and MTT assay for HeLa cells is shown (b) The sensitivity of the developed approach using either DAPI or Hoechst 33342 and MTT assay for late-passage IMR-90 cells is shown (a) A comparison of the results of cytotoxicity evaluation of cytarabine and TFdU for HeLa cells provided by the developed approach based on DAPI or Hoechst 33342 or by MTT assay is shown (b) A comparison of the results of cytotoxicity evaluation of TFdU for NCI-H460 and NCI-H2009 cells and HmdU for NCI-H2009 cells provided by the developed approach based on DAPI or by MTT assay is shown The possibility to perform the detection of various components and cell cycle analysis simultaneously with the cell quantification allows various analyses to be conducted using the same cells and to address e.g the effect of the cytotoxicity of various compounds in a more complex way Various assays for the measurement of cell proliferation are presently available The most used approaches exploit two basic strategies The first is based on the measurement of the enzyme activities The second is based on the determination of the amount of nucleic acids The enzyme activities in cell samples are most commonly measured by MTT The developed approach does not depend on the cell line and is definitely shorter than 2 hours including the time necessary for the data collection It can be also interrupted for at least 3 months (after fixation) or for at least 20 days (after elution) Another disadvantage of metabolic assays is the necessity to add the substrates under sterile conditions as cells are usually returned to the incubators There is no need for sterility in the case of the developed approach we found that the presence of SDS results in around a 90-fold increase of the fluorescence of DAPI a 700-fold increase in the case of Hoechst 333258 and around a 1,000-fold signal increase in the case of Hoechst 33342 the increase of the fluorescence intensity is not so important in the developed assay the absolute fluorescence intensity plays the most important role as only the dye eluted from the cells is analysed Hoechst 33258 provided only 75% of the signal of Hoechst 33342 while DAPI provided only 45% of this signal Although a variant of the CyQuant approach based on cell permeabilisation without a freezing step can be used it provides the linear data from 100 to 20,000 cells only the developed approach provides linear data from around 70 HeLa cells to the fully confluent cell population (ca 71,000 HeLa cells per well of a 96-well plate) without the need of repeated sample freezing/thawing and cell lysis if DAPI is used Even higher sensitivity and linearity (35 HeLa cells or 50-70 human diploid fibroblasts per well of a 96-well plate) can be obtained if Hoechst 33342 is used the maximum number of cells should correspond to the around 35,500 cells per well of a 96-well plate (around 50% confluence) The cell lysis excludes the simultaneous analysis of the cell cycle by image cytometry or simultaneous analysis of the cell population by immunocytochemical methods the method developed does not require cell lysis As the approach is compatible with the commonly used ethanol fixation both the cell cycle analysis using image cytometry or immunocytochemical detections can be performed before cell quantification DAPI and Hoechst staining is fully compatible with the commonly used fluorochromes as their excitation and emission spectra are sufficiently separated from the spectra of DAPI and Hoechst dyes The cells fixed by formaldehyde can be also used during the developed assay as the formaldehyde fixation provides approximately half of the signal obtained after ethanol fixation Another advantage of the developed method is the possibility of repeated staining of the cells if necessary (e.g the developed approach provided very similar data as the MTT assay despite the fact that that it provides much higher linearity It is probably the result of the very steep decline of the survival of the cells if fivefold dilution of the tested substances is used We suppose that the presented procedure can also be highly valuable if metabolically less active adherent cells are processed and the low sensitivity of metabolic assays therefore makes it impossible to obtain the relevant data the method developed provides relative data by means of a haemocytometer or cameras and suitable software it is possible to determine the absolute number of cells in the particular sample The described method of cell quantification is fast and thanks to the low material demands and the fact that the measurement takes place in another vessel than the one in which the cells are cultivated it is suitable for processing high numbers of samples and/or large samples the cost of the chemicals used for the processing of one 96-well plate is around 1.5 USD Although the price of the black well plate increases the expenditures the two washing steps with 0.2 M KOH or the washing buffer used for Hoechst dyes for 30 minutes followed by two washing steps in tap water and one washing step with deionized water provided similar results as a new black well plate The developed method of cell quantification is based on the measurement of the DNA content In contrast to commonly used methods based on the same strategy it does not rely on the enhancement of the signal by binding the dye to nucleic acids Hoechst or DAPI dyes bound to DNA are eluted using an elution buffer and the enhancement of the signal is performed in this buffer by SDS The formation of micelles of SDS results in a high enhancement of the fluorescence of the used dyes (e.g up to a 1,000-fold increase of the fluorescence intensity in the case of Hoechst 33342) The signal is high for at least 20 days at RT The procedure can be also interrupted for several months after ethanol fixation the cell cycle analysis using image cytometry or the detection of various cellular components can be performed before the elution step For the samples with a cell density below 50% confluence It makes it possible to obtain high linearity and sensitivity If the samples with cells at a higher density are used The method can be also used for cytotoxicity evaluation carcinoma; both latter cell lines are a gift from Dr Institute of Molecular and Translational Medicine human diploid fibroblasts: IMR-90 cells (lung CCL-186) and MRC-5 cells (lung; a gift from Dr NCI-H2009 and NCI-H460 cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum 3.7 g/l of sodium bicarbonate and 50 µg/ml of gentamicin The IMR-90 and MRC-5 cells were cultivated in Eagle’s minimum essential medium (EMEM) supplemented with 20% foetal bovine serum The cells were cultivated in culture flasks or in a Petri dishes or in 96-well plates at 37 °C in a humidified atmosphere containing 5% CO2 the cells were seeded at the density of 5 × 103 cells per well in 96-well plates and incubated for 24 hours the modified nucleosides were added to the culture media Serial fivefold dilutions of cytarabine starting at 0.00064 µM and ending at 50 µM and serial fivefold dilutions of TFdU or HmdU starting at 0.0032 µM and ending at 250 µM were used The cells were further incubated for the time corresponding to the two population doubling times (HeLa cells and NCI-H460 – 48 hours the culture media were exchanged for nucleoside-free media and the cells were incubated for an additional three population doubling times (HeLa cells and NCI-H460 – 72 hours 1.1 mM MTT was added and the same protocol was used as in the case of the linearity testing or the plates were processed according to the developed approach The cells were rinsed with 1× PBS and fixed with 70% ethanol or 100% methanol for 10 minutes or 30 minutes or 1 hour or overnight at −20 °C or 0 °C or at RT the culture medium was removed from the culture dish and the fixative was added directly to the cells without the previous washing with the buffer the cells fixed in 70% ethanol were stored for 3 months at −20 °C the cells were rinsed with 1× PBS and 2% formaldehyde in 1× PBS was added to the samples for 10 minutes at RT the cells were further permeabilised with 0.2% Triton X-100 in 1x PBS for 10 minutes the buffer was removed and the cells were air dried The 1 µM solutions of DAPI or Hoechst dyes in water or in 20 mM phosphate buffer pH 7 containing various concentrations of SDS or without SDS were prepared The 0.1 ml of the prepared solutions was then transferred into the black 96-well plate and the fluorescence was measured using the plate reader The buffered solutions of 1 µM DAPI or Hoechst dye 33342 containing 8% 0.125% and 0.3125% SDS were used during the optimization of the SDS concentrations For the analysis of the effect of micelle formation on the signal intensity a 1 µM solution of DAPI or Hoechst dyes in water containing 0.5% 0.0078125% or 0.00390625% SDS was prepared then 0.1 ml of the solution was transferred into the black 96-well plate and the fluorescence was measured The excitation and the emission spectra of DAPI were measured using a 1 µM solution of DAPI in 20 mM Tris–HCl The excitation and the emission spectra of Hoechst 33342 and 33258 were measured using a 1 µM solution of Hoechst dyes in a 20 mM phosphate buffer the HeLa cells were fixed with 70% ethanol for 10 minutes at RT air-dried and incubated with the solution of RNase A The final concentration of RNase A was 0.1 mg/ml The samples were incubated 60 minutes at 37 °C and 300 rpm the samples were processed according to the developed protocol the signal was measured using the Infinite 200 Pro Plate Reader (Tecan) If the image cytometry or manual counting was used for the sample evaluation the images were obtained by an Olympus IX81 microscope (objective: UPLFLN 10 × NA 0.3) equipped with a Hamamatsu ORCA II camera (a resolution of 1344 × 1024 pixels) using Cell ∧ R acquisition software The data were analysed using CellProfiler and Microsoft Excel and the final graphs were made in GraphPad Prism 6 The graphs were constructed using the following functions: Standard four parameter logistic nonlinear regression was used in the case of the analysis of the standard cytotoxic test by MTT assay or by the developed approach One-phase association was used for the analysis of the signal dependence on the number of HeLa cells in the case of MTT assay Linear regression or second order polynomial (quadratic) regression was used for the analysis of the signal dependence on the concentration of the DNA dyes diluted in 2% SDS the signal dependence on the number of HeLa cells IMR-90 cells and MRC-5 in the case of Hoechst 33342 All the performed experiments were done in three independent replicates The data are presented as the mean values ± standard deviation (SD) The Fig. 1 was done using Rhinoceros 5 and Adobe Photoshop CS4 software All data generated or analysed during this study are included in this published article (and its Supplementary Information files) Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays MTS interferon assay: A simplified cellular dehydrogenase assay for interferon activity using a water-soluble tetrazolium salt A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1) Evaluation of a Soluble Tetrazolium Formazan Assay for Cell-Growth and Drug Sensitivity in Culture Using Human and Other Tumor-Cell Lines Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity Evaluation of the alamarblue assay for adherent cell irradiation experiments The challenge to measure cell proliferation in two and three dimensions Sensitive determination of cell number using the CyQUANT (R) cell proliferation assay Direct measurement of cell numbers in microtitre plate cultures using the fluorescent dye SYBR green I Measurement of cell numbers in microtiter culture plates using the fluorescent dye Hoechst 33258 A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro Interaction of Small Molecules with Double-Stranded RNA: Spectroscopic and Calorimetric Study of Hoechst and Proflavine Binding to PolyCG Structures The Search for Structure-Specific Nucleic-Acid Interactive Drugs - Effects of Compound Structure on Rna Versus DNA Interaction Strength Dapi (4′,6-Diamidino-2-Phenylindole) Binds Differently to DNA and Rna - Minor-Groove Binding at at Sites and Intercalation at Au Sites Fluorescent complexes of DNA with DAPI 4′,6-diamidine-2-phenyl indole.2HCl or DCI 4′,6-dicarboxyamide-2-phenyl indole Direct and sensitized photoprocesses of bis-benzimidazole dyes and the effects of surfactants and DNA Determination of critical micelle concentration values using capillary electrophoresis instrumentation The Genomic and Transcriptomic Landscape of a HeLa Cell Line Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping Cell Proliferation and Cytotoxicity Assays cytotoxic effect and cell-cycle perturbation of Hoechst 33342 on L1210 cells in vitro Intracellular fluorescent labelling of cells for analysis of lymphocyte migration Vitality measurement using spectrum shift in Hoechst 33342 stained cells DNA stainability in aneuploid breast tumors: comparison of four DNA fluorochromes differing in binding properties Development of an ultrasensitive in vitro assay to monitor growth of primary cell cultures with reduced mitotic activity Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA Photophysical properties of fluorescent DNA-dyes bound to single- and double-stranded DNA in aqueous buffered solution Pdr18 is involved in yeast response to acetic acid stress counteracting the decrease of plasma membrane ergosterol content and order Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation Ligasová, A. et al. Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine. Open Biol. 6, 150172, https://doi.org/10.1098/rsob.150172 (2016) Ligasová, A., Strunin, D., Friedecký, D., Adam, T. & Koberna, K. A fatal combination: a thymidylate synthase inhibitor with DNA damaging activity. PLoS One 10, e0117459, https://doi.org/10.1371/journal.pone.0117459 (2015) CellProfiler: image analysis software for identifying and quantifying cell phenotypes function and compatibility for CellProfiler: modular high-throughput image analysis software Ligasová, A., Strunin, D., Liboska, R., Rosenberg, I. & Koberna, K. Atomic Scissors: A New Method of Tracking the 5-Bromo-2 ‘-Deoxyuridine-Labeled DNA In Situ. Plos One 7, e52584, https://doi.org/10.1371/journal.pone.0052584 (2012) Download references This work was supported by the Ministry of Health of the Czech Republic [15-31604A]; the Ministry of Education Youth and Sports of the Czech Republic - Project EATRIS-CZ [LM2015064]; and the European Regional Development Fund - Project ENOCH [CZ.02.1.01/0.0/0.0/16_019/0000868] Palacký University Olomouc holds a Czech patent (307415) and has pending a European patent application (EP 18169749.1) for method for cell quantification Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-019-45217-9 Applied Biochemistry and Biotechnology (2025) Metrics details An Author Correction to this article was published on 03 September 2021 This article has been updated nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing Because nuclear condensation and fragmentation can be typical markers of cell death we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes Despite the unique Hoechst probe properties for nuclear changes detection reported a quantitative spectrofluorometric method development the aim of the present study was to develop a Hoechst 33258 dye spectrofluorometric assay for quantitative measurement of nuclear condensation and fragmentation in intact cells we aimed to evaluate the obtained outcomes in comparison with other frequently used methods for cell damage (i.e WST-1 and glutathione assays) and apoptosis (TUNEL Optimization of the spectrofluorometric assay for detection of nuclear condensation and fragmentation in HepG2 cells (A) Optimization of H33258 concentration (0.1; 1; 2; 5 µg/mL) – untreated cells (white columns) 100 µM cisplatin treated cells (blue columns) (B) Change of intensity of fluorescence over time HepG2 cells were treated with CisPt for 24 h the cells were incubated with H33258 (2 µg/mL) and fluorescence (EX/EM = 352/461 nm) was recorded for 10 min (C,D) Detection of nuclear condensation and fragmentation in cells HepG2 cells were treated with cisplatin for 24 h the cells were incubated with H33258 (2 µg/mL) and fluorescence (EX/EM = 352/461 nm) was recorded for 5 min the background fluorescence was subtracted and fluorescence intensity corresponding to the extent of nuclear condensation and fragmentation was showed in untreated (white) 50 µM (light blue) and 100 µM CisPt (dark blue) treated cells (= C) The fluorescence intensity attributed to the extent of nuclear condensation and fragmentation was compared among untreated cells (white) 100 µM CisPt (dark blue) treated cells (= D) For estimation of optimal cell incubation time with H33258 HepG2 cells were treated with CisPt (0; 50; 100 µM) for 24 h We found that centrifugation of cells (5 min 8000g; RT) after the treatment with tested compounds followed by cell culture medium replacement is crucial for achieving repeatable results because it ensures the sedimentation of all cells on the bottom of a well 70 µL of culture medium was replaced with 70 µL of PBS 1 × in each well 10 µL of H33258 was added to obtain final concentration 2 µg/mL H33258 in a well and fluorescence was recorded at EX/EM = 352/461 nm Characterizing cisplatin-induced toxicity in HepG2 and HK-2 cells HepG2 (A,C,E) and HK-2 (B,D,F) cells were incubated with cisplatin (CisPt; 0; 0.5; 5; 25 and 100 µM) for 24 and 48 h nuclear condensation and fragmentation using the H33258 spectrofluorometric assay (A,B) dehydrogenase activity using the WST-1 test (C,D) and glutathione levels using monochlorobimane assay (E,F) were measured (*p < 0.05; **p < 0.01; ***p < 0.001 untreated cells at appropriate time interval) while for HepG2 the significant reduction was ≥ 25 µM both cell types had significant reduction in similar CisPt concentrations treatments ≥ 5 µM the outcomes found using the WST-1 and glutathione assays confirmed the occurrence of CisPt toxicity detected using the H33258 spectrofluorometric assay WST-1 and glutathione assays detected cellular damage also in cells treated with 5 µM CisPt both biochemical assays are more sensitive in detection of a cell damage in comparison to H33258 assay detecting structural nuclear changes Detection of nuclear condensation and fragmentation using the H33258 spectrofluorometric assay HepG2 (A) and HK-2 (B) cells were treated with cisplatin (CisPt The cells treated with TiO2 P25 nanoparticles (NPs orange columns) were used as a negative control the cells were incubated with H33258 (2 µg/mL) for 5 min and intensity fluorescence (EX/EM = 352/461 nm) was measured the background fluorescence was subtracted and the extent of nuclear condensation was expressed in RFU Data are presented as mean ± SEM (n = 8–20) (*p < 0.05; **p < 0.01; ***p < 0.001; vs To evaluate the outcomes of newly developed H33258 spectrofluorometric assay we aimed to compare the results with other methods characterizing pro-apoptotic processes in cells we treated HepG2 and HK-2 cells with CisPt (50 and 100 µM) for 6 24 and 48 h and characterized nuclear changes and pro-apoptotic activation using four additional measurements Caspase 3/7 activity in CisPt treated cells HepG2 (A) and HK-2 (B) cells were treated with CisPt (50 and 100 µM) for 6 the activity of caspases 3/7 was measured (EX/EM = 485/535 nm) Phosphorylation of JNK1 and JNK2 can also correspond with activated apoptotic process and that is why we aimed to estimate their levels We observed that pJNK1 levels were increased in all tested time incubation with CisPt in HepG2 cells protein expressions of pJNK1 and pJNK2 were stimulated predominantly at 24 h of treatment the detected increase of pJNK levels correlated strongly with PARP fragmentation in both cell lines HepG2 and HK-2 cells were treated with CisPt (50 and 100 µM) for 6 the cells and nuclei were visualized using phase contrast (left TUNEL (FITC 480/30 nm; middle) and Hoechst 33258 stainings (DAPI 375/28 nm; right) DNA ladder assay in CisPt treated cells. HepG2 (A) and HK-2 (B) cells were treated with CisPt (50 and 100 µM) for 6, 24 and 48 h. After DNA isolation, DNA ladder assay was performed. (0, untreated cells; 50, 50 µM CisPt; 100, 100 µM CisPt; M, 100 bp marker). Original gels images were included into the Supplementary file these reports used H33258 only for microscopic detection of nuclear changes we aimed to develop a quantitative spectrofluorometric method for the detection of nuclear condensation and fragmentation using H33258 in cultured cells we decided to estimate H33258 concentrations in the range of 0.1–5 µg/mL for development of the spectrofluorometric assay We selected 2 µg/mL H33258 to be optimal for treatment of cells without risk of cell impairment during the spectrofluorometric measurement for 5 min the newly developed H33258 spectrofluorometric method provided similar outcomes to other scientific reports studying apoptosis induced by camptothecin we can conclude again that the spectrofluorometric method using H33258 provided results similar to the outcomes of other studies characterizing staurosporine action in cells our findings of increased nuclear condensation and fragmentation in 50 and 100 µM CisPt treated HepG2 and HK-2 cells using the developed spectrofluorometric assay are fully consistent with the literature We summarize that the findings of DNA fragmentation and nuclear condensation detected using all the biochemical assays in HepG2 and HK-2 cells after cisplatin treatment presented in this study are in accordance to published reports of other authors testing cisplatin as well We developed a spectrofluorometric method using Hoechst 33258 staining which detects nuclear condensation and fragmentation in intact cells Our results showed that the spectrofluorometric method was capable to detect the nuclear changes in three typical pro-apoptotic agents we compared the results obtained using the spectrofluorometric assay with outcomes of other methods characterizing apoptotic processes we conclude that here developed spectrofluorometric method is capable to detect nuclear pro-apoptotic changes of similar sensitivity and specificity to that of caspase 3/7 activity measurement and TUNEL assay we suppose that the spectrofluorimetric H33258 method could join other routinely used methods characterizing apoptosis in cells the developed spectrofluorometric assay possesses several advantages (e.g low-cost) implying its potential use in assessing nuclear condensation and fragmentation in routine laboratory practice and in high-throughput screening studies 1 mg/mL) and all other chemicals (formaldehyde TiO2 P25) were purchased from Sigma Aldrich All materials for cell culture were purchased from Sigma-Aldrich (USA) if not otherwise specified a human hepatocellular carcinoma cell line HepG2 cells were cultured in Dulbecco's Modified Eagle's Medium with high glucose content (4500 g/L w/wo phenol red) supplemented with 10% (v/v) fetal bovine serum All the experiments were conducted using HepG2 cells in passages 4–15 All the experiments were conducted using the HK-2 cells in passages 5–15 HepG2 and HK-2 cells were tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza) All cells used in the experiments were mycoplasma free DNA fingerprinting) was used for HepG2 and HK-2 cell line authentication using a commercial kit in Generi Biotech (Czech Republic) The STR analysis proved 100% conformity of both HepG2 and HK-2 cells with the reference standards The HepG2 and HK-2 cells were seeded in 100 µL of appropriate cell culture medium in 96-well plates at density of 1.5 × 104 and 2 × 104 cells per well for 24 h (if not stated otherwise) staurosporine (STA; 0–100 nM) and TiO2 P25 nanoparticles (NPs; 0–10 µg/mL) All compounds were diluted in appropriate cell culture mediums to obtain final concentrations the culture medium was replaced by 100 µL of medium containing a tested compound and the cells were treated for 6 To characterize the extent of cell impairment we used the newly developed spectrofluorometric assay using H33258 together with caspase activity and proteins expression measurements To develop a spectrofluorometric method for detection of changes in nuclear condensation and fragmentation in intact cells we used HepG2 and HK-2 cells at confluency 50–70% After treatment with tested compounds (CisPt the cells grown in a 96-well plate were centrifuged (5 min 70 µL of a supernatant was replaced with 70 µL of warmed phosphate-buffered saline (PBS 1 × 37 °C) and 10 µL of H33258 solution (in PBS 1 ×) was added to a well The final concentrations of H33258 in a well were 0.1–5 µg/mL the cells were incubated with H33258 for 60 min during optimization of the assay or for 5 min at optimal conditions and the spectrofluorometric measurement was performed at EX/EM = 352/461 nm (EX/EM slit widths 25/25 nm) using a Tecan Spark fluorescence microplate reader (Tecan The samples were measured at least in triplicates the fluorescence signal was presented in Relative Fluorescence Units (RFU) as mean ± SEM All spectrofluorometric measurements presented here were repeated at least in three independent experiments Dehydrogenase activity was evaluated by WST-1 test (Roche The WST-1 test measures the activity of intra- and extramitochondrial dehydrogenases 10 µL of WST-1 reagent was added to the treated cells The absorbance change (0–1 h) was measured spectrophotometrically at wavelength of 440 nm using a Tecan Spark fluorescence microplate reader (Tecan The cell viability was expressed as the percentage of intra- and extramitochondrial dehydrogenases activity relative to that in control cells (= 100%) 20 µL of the bimane solution was added to cells to obtain the final concentration 40 µM and spectrofluorometric measurement was started immediately The fluorescence increase (EX/EM = 394/490 nm) was measured for 10 min using a Tecan Spark fluorescence microplate reader (Tecan The fluorescence was expressed as the slope of change in fluorescence over time The glutathione levels were expressed as the percentage relative to the glutathione levels in control cells (= 100%) Caspase 3/7 activity in HepG2 and HK-2 cells was detected using ApoONE Homogeneous Caspase-3/7 Assay (Promega USA) according to the manufacturer´s instructions 100 µL of the caspase 3/7 working solution was added to treated cells the fluorescence (EX/EM = 485/535 nm) was measured in duplicates using a Tecan Spark fluorescence microplate reader (Tecan The caspase 3/7 activity levels were expressed as the percentage relative to the caspase 3/7 activity levels in control cells (= 100%) Capillary Western Immunoassay was performed in protein lysates from HepG2 and HK-2 cells cultured in 6-well plates at density of 5 × 105 and 1.3 × 106 cells per well the culture medium was replaced by 2 mL of CisPt solution and the cells were treated for 6 the cells were washed twice with PBS 1 × and protein lysates were prepared by lysing cells with 400 µL of RIPA buffer (Sigma-Aldrich USA) with MS-safe protease and phosphatase inhibitor (Sigma-Aldrich Capillary Western Immunoassay was performed according to manufacturer´s instructions (Protein Simple protein lysates were analyzed on a Wes system (ProteinSimple USA) using a 12–230 kDa Separation Module (Biotechne poly-(ADP-ribose) polymerase-1 (PARP-1; primary antibody 1:100; Cell Signalling USA) were normalized using the reference protein β-actin (primary antibody 1:500 The peaks were analyzed using Compass software (Protein Simple Two criteria were used for the discrimination of signals from the background: (1) the peak high must be higher or equal to 1000 and (2) the peak’s signal-to-noise ratio given by the software must be higher or equal to 10 The results were counted as:$\frac{{{\text{area}}\,{\text{of}}\,{\text{the}}\,{\text{peak}}\,{\text{of}}\,{\text{interest}}}}{{{\text{area}}\,{\text{of}}\,{\text{the}}\,{\text{peak}}\,{\text{of}}\,\upbeta {\text{ - actin}}}}$ TUNEL assay followed by H33258 staining of nuclei was performed in HepG2 and HK-2 cells cultured in 200 µL of appropriate cell culture medium on cell culture chamber slides at density of 1.5 × 105 and 2 × 105 cells per well the culture medium was replaced by 200 µL of CisPt solutions and the cells were treated for 6 TUNEL assay was performed using Click-iT TUNEL Alexa Fluor 488 Imaging Assay kit (ThermoFisher Scientific USA) according to manufacturer´s instructions The cells were fixed with 12% formaldehyde for 15 min at 37 °C the cells were permeabilized with 0.2% Triton X-100 for 15 min at 37 °C washed with PBS 1 × and incubated with terminal deoxynucleotidyl transferase (TdT) buffer for 10 min at 37 °C cells were mixed with a TdT reaction mixture (TdT buffer the cells were washed with 3% bovine serum albumin (BSA) and Click IT reagent for fluorescent staining was added for 30 min at 37 °C H33258 at a final concentration of 2 µg/mL was used to visualize the cell nuclei 375/28 nm) were visualized with an Eclipse 80i fluorescence microscope (Nikon DNA ladder was performed in HepG2 and HK-2 cells cultured in 6-well plates at density of 5 × 105 and 1 × 106 cells per well the culture medium was replaced by 2 mL of CisPt and the cells were treated for 6 DNA was isolated from treated cells using The ApoTarget Quick Apoptotic DNA Ladder Detection Kit (Invitrogen Isolated DNA samples were loaded onto a 1.5% agarose gel with 0.5 mg/mL ethidium bromide (Top-Bio Czech Republic) followed by electrophoresis (5 V/cm) DNA was visualized by an ultraviolet gel documentation system (Vilber Lourmat GeneRuler 100 bp DNA ladder (ThermoFisher Scientific Statistical analysis was performed using OriginPro 9.0.0 (OriginLab Statistical significance was analyzed after normality testing using one-way analysis of variance (ANOVA) followed by Tukey’s test at significance level p = 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001) A Correction to this paper has been published: https://doi.org/10.1038/s41598-021-97792-5 Wyllie, A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555–556. https://doi.org/10.1038/284555a0 (1980) Elmore, S. Apoptosis: A review of programmed cell death. Toxicol. Pathol. 35, 495–516. https://doi.org/10.1080/01926230701320337 (2007) Caruso, S. et al. Defining the role of cytoskeletal components in the formation of apoptopodia and apoptotic bodies during apoptosis. Apoptosis 24, 862–877. https://doi.org/10.1007/s10495-019-01565-5 (2019) Hou, L. et al. Necrotic pyknosis is a morphologically and biochemically distinct event from apoptotic pyknosis. J. Cell Sci. 129, 3084–3090. https://doi.org/10.1242/jcs.184374 (2016) Burgoyne, L. A. The mechanisms of pyknosis: Hypercondensation and death. Exp. Cell Res. 248, 214–222. https://doi.org/10.1006/excr.1999.4406 (1999) Gotzmann, J., Meissner, M. & Gerner, C. The fate of the nuclear matrix-associated-region-binding protein SATB1 during apoptosis. Cell Death Differ. 7, 425–438. https://doi.org/10.1038/sj.cdd.4400668 (2000) Takada, S., Watanabe, T. & Mizuta, R. DNase gamma-dependent DNA fragmentation causes karyolysis in necrotic hepatocyte. J. Vet. Med. Sci. 82, 23–26. https://doi.org/10.1292/jvms.19-0499 (2020) Ferri, K. F. & Kroemer, G. Control of apoptotic DNA degradation. Nat. Cell Biol. 2, E63-64. https://doi.org/10.1038/35008692 (2000) Walsh, J. G. et al. Executioner caspase-3 and caspase-7 are functionally distinct proteases. Proc. Natl. Acad. Sci. USA 105, 12815–12819. https://doi.org/10.1073/pnas.0707715105 (2008) Zhou, P., Lugovskoy, A. A., McCarty, J. S., Li, P. & Wagner, G. Solution structure of DFF40 and DFF45 N-terminal domain complex and mutual chaperone activity of DFF40 and DFF45. Proc. Natl. Acad. Sci. USA 98, 6051–6055. https://doi.org/10.1073/pnas.111145098 (2001) Nagata, S., Nagase, H., Kawane, K., Mukae, N. & Fukuyama, H. Degradation of chromosomal DNA during apoptosis. Cell Death Differ. 10, 108–116. https://doi.org/10.1038/sj.cdd.4401161 (2003) Skalka, M., Matyasova, J. & Cejkova, M. Dna in chromatin of irradiated lymphoid tissues degrades in vivo into regular fragments. FEBS Lett. 72, 271–274. https://doi.org/10.1016/0014-5793(76)80984-2 (1976) Pariente, R., Pariente, J. A., Rodríguez, A. B. & Espino, J. Melatonin sensitizes human cervical cancer HeLa cells to cisplatin-induced cytotoxicity and apoptosis: Effects on oxidative stress and DNA fragmentation. J. Pineal Res. 60, 55–64. https://doi.org/10.1111/jpi.12288 (2016) Majtnerová, P. & Roušar, T. An overview of apoptosis assays detecting DNA fragmentation. Mol. Biol. Rep. 45, 1469–1478. https://doi.org/10.1007/s11033-018-4258-9 (2018) Martin, R. M., Leonhardt, H. & Cardoso, M. C. DNA labeling in living cells. Cytometry A 67, 45–52. https://doi.org/10.1002/cyto.a.20172 (2005) Zhang, X. T., Song, T. B., Du, B. L., Li, D. M. & Li, X. M. Caspase-3 antisense oligodeoxynucleotides inhibit apoptosis in gamma-irradiated human leukemia HL-60 cells. Apoptosis 12, 743–751. https://doi.org/10.1007/s10495-006-0018-8 (2007) Miao, Z. H. et al. 4-nitroquinoline-1-oxide induces the formation of cellular topoisomerase I-DNA cleavage complexes. Cancer Res. 66, 6540–6545. https://doi.org/10.1158/0008-5472.CAN-05-4471 (2006) Replication checkpoint selectivity for late S phase cells induced by topoisomerase I cleavage complexes Gomes, C. J., Harman, M. W., Centuori, S. M., Wolgemuth, C. W. & Martinez, J. D. Measuring DNA content in live cells by fluorescence microscopy. Cell Div. 13, 6. https://doi.org/10.1186/s13008-018-0039-z (2018) Liu, C.-H., Tsao, M.-H., Sahoo, S. L. & Wu, W.-C. Magnetic nanoparticles with fluorescence and affinity for DNA sensing and nucleus staining. RSC Adv. 7, 5937–5947. https://doi.org/10.1039/c6ra25610d (2017) Campos, V., Rappaz, B., Kuttler, F., Turcatti, G. & Naveiras, O. High-throughput, nonperturbing quantification of lipid droplets with digital holographic microscopy. J. Lipid Res. 59, 1301–1310. https://doi.org/10.1194/jlr.D085217 (2018) Nogueira, E. et al. Assessment of liposome disruption to quantify drug delivery in vitro. Biochim. Biophys. Acta 1858, 163–167. https://doi.org/10.1016/j.bbamem.2015.11.008 (2016) Zhou, Y. B. et al. Multiplexed imaging detection of live cell intracellular changes in early apoptosis with aggregation-induced emission fluorogens. Sci. China Chem. 61, 892–897. https://doi.org/10.1007/s11426-018-9287-x (2018) Sakamoto, T., Hasegawa, D. & Fujimoto, K. Fluorine-modified bisbenzimide derivative as a molecular probe for bimodal and simultaneous detection of DNAs by F-19 NMR and fluorescence. Chem. Commun. 51, 8749–8752. https://doi.org/10.1039/c5cc01995h (2015) Martin, R. M., Leonhardt, H. & Cardoso, M. C. DNA labeling in living cells. Cytom. Part A 67A, 45–52. https://doi.org/10.1002/cyto.a.20172 (2005) Qin, G. Q. et al. Dihydroartemisinin induces apoptosis preferentially via a Bim-mediated intrinsic pathway in hepatocarcinoma cells. Apoptosis 20, 1072–1086. https://doi.org/10.1007/s10495-015-1132-2 (2015) Kapoor, R., Rizvi, F. & Kakkar, P. Naringenin prevents high glucose-induced mitochondria-mediated apoptosis involving AIF, Endo-G and caspases. Apoptosis 18, 9–27. https://doi.org/10.1007/s10495-012-0781-7 (2013) Chen, G. et al. Mammalian target of rapamycin regulates isoliquiritigenin-induced autophagic and apoptotic cell death in adenoid cystic carcinoma cells. Apoptosis 17, 90–101. https://doi.org/10.1007/s10495-011-0658-1 (2012) Soares, J. et al. Structure-cytotoxicity relationship profile of 13 synthetic cathinones in differentiated human SH-SY5Y neuronal cells. Neurotoxicology 75, 158–173. https://doi.org/10.1016/j.neuro.2019.08.009 (2019) Zhang, Z. et al. Cytochrome P450 2A13 is an efficient enzyme in metabolic activation of aflatoxin G1 in human bronchial epithelial cells. Arch. Toxicol. 87, 1697–1707. https://doi.org/10.1007/s00204-013-1108-3 (2013) Wang, J. et al. Biochanin A protects against lipopolysaccharide-induced damage of dopaminergic neurons both in vivo and in vitro via inhibition of microglial activation. Neurotox. Res. 30, 486–498. https://doi.org/10.1007/s12640-016-9648-y (2016) Ki, S. H. et al. Artificial vesicles as an animal cell model for the study of biological application of non-thermal plasma. J. Phys. D Appl. Phys. 49, Artn 085401 https://doi.org/10.1088/0022-3727/49/8/085401 (2016) Aston, N. S., Watt, N., Morton, I. E., Tanner, M. S. & Evans, G. S. Copper toxicity affects proliferation and viability of human hepatoma cells (HepG2 line). Hum. Exp. Toxicol. 19, 367–376. https://doi.org/10.1191/096032700678815963 (2000) Handl, J., Capek, J., Majtnerova, P., Bacova, J. & Rousar, T. The effect of repeated passaging on the susceptibility of human proximal tubular HK-2 cells to toxic compounds. Physiol. Res. 69, 731–738. https://doi.org/10.33549/physiolres.934491 (2020) Cell cycle specific toxicity of the Hoechst 33342 stain in untreated or irradiated murine tumor cells Purschke, M., Rubio, N., Held, K. D. & Redmond, R. W. Phototoxicity of Hoechst 33342 in time-lapse fluorescence microscopy. Photochem. Photobiol. Sci. 9, 1634–1639. https://doi.org/10.1039/c0pp00234h (2010) Hammond, C. L., Madejczyk, M. S. & Ballatori, N. Activation of plasma membrane reduced glutathione transport in death receptor apoptosis of HepG2 cells. Toxicol. Appl. Pharmacol. 195, 12–22. https://doi.org/10.1016/j.taap.2003.10.008 (2004) Cao, M. R. et al. Harmine induces apoptosis in HepG2 cells via mitochondrial signaling pathway. Hepatobiliary Pancreat. Dis. Int. 10, 599–604. https://doi.org/10.1016/s1499-3872(11)60102-1 (2011) Beaton-Green, L. A. & Wilkins, R. C. Quantitation of chromosome damage by imaging flow cytometry. Methods Mol. Biol. 1389, 97–110. https://doi.org/10.1007/978-1-4939-3302-0_6 (2016) Bogush, T. A. et al. A new approach to epithelial-mesenchymal transition diagnostics in epithelial tumors: Double immunofluorescent staining and flow cytometry. Biotechniques https://doi.org/10.2144/btn-2020-0024 (2020) Zhang, L. J., Li, Z. Q., Yang, Y. P., Li, X. W. & Ji, J. F. Tunicamycin suppresses cisplatin-induced HepG2 cell apoptosis via enhancing p53 protein nuclear export. Mol. Cell. Biochem. 327, 171–182. https://doi.org/10.1007/s11010-009-0055-z (2009) Yang, S. K. et al. Mitochondria targeted peptide SS-31 prevent on cisplatin-induced acute kidney injury via regulating mitochondrial ROS-NLRP3 pathway. Biomed. Pharmacother. 130, 110521. https://doi.org/10.1016/j.biopha.2020.110521 (2020) Tanida, S. et al. Mechanisms of cisplatin-induced apoptosis and of cisplatin sensitivity: Potential of BIN1 to act as a potent predictor of cisplatin sensitivity in gastric cancer treatment. Int. J. Surg. Oncol. 2012, 862879. https://doi.org/10.1155/2012/862879 (2012) Garcia, C. P. et al. Topoisomerase I inhibitor, camptothecin, induces apoptogenic signaling in human embryonic stem cells. Stem Cell Res. 12, 400–414. https://doi.org/10.1016/j.scr.2013.12.002 (2014) Rath, G. et al. De novo ceramide synthesis is responsible for the anti-tumor properties of camptothecin and doxorubicin in follicular thyroid carcinoma. Int. J. Biochem. Cell B 41, 1165–1172. https://doi.org/10.1016/j.biocel.2008.10.021 (2009) Pommier, Y. Topoisomerase I inhibitors: Camptothecins and beyond. Nat. Rev. Cancer 6, 789–802. https://doi.org/10.1038/Nrc1977 (2006) Ding, Y. M., Wang, B., Chen, X. Y., Zhou, Y. & Ge, J. H. Staurosporine suppresses survival of HepG2 cancer cells through Omi/HtrA2-mediated inhibition of PI3K/Akt signaling pathway. Tumor Biol. 39, Artn 694317 https://doi.org/10.1177/1010428317694317 (2017) Deshmukh, M. & Johnson, E. M. Staurosporine-induced neuronal death: Multiple mechanisms and methodological implications. Cell Death Differ. 7, 250–261. https://doi.org/10.1038/sj.cdd.4400641 (2000) Petkovic, J. et al. DNA damage and alterations in expression of DNA damage responsive genes induced by TiO2 nanoparticles in human hepatoma HepG2 cells. Nanotoxicology 5, 341–353. https://doi.org/10.3109/17435390.2010.507316 (2011) Ji, Y. B. & Gao, S. Y. Arylamine N-acetyltransferases: a new inhibitor of apoptosis in HepG2 cells. J. Zhejiang Univ.-Sc B 9, 701–706. https://doi.org/10.1631/jzus.B0820090 (2008) Gorman, A. M., Hirt, U. A., Orrenius, S. & Ceccatelli, S. Dexamethasone pre-treatment interferes with apoptotic death in glioma cells. Neuroscience 96, 417–425. https://doi.org/10.1016/S0306-4522(99)00565-5 (2000) Cisplatin induces apoptosis in LLC-PK1 cells via activation of mitochondrial pathways Schweyer, S. et al. Cisplatin-induced apoptosis in human malignant testicular germ cell lines depends on MEK/ERK activation. Br. J. Cancer 91, 589–598. https://doi.org/10.1038/sj.bjc.6601919 (2004) Rathinam, R., Ghosh, S., Neumann, W. L. & Jamesdaniel, S. Cisplatin-induced apoptosis in auditory, renal, and neuronal cells is associated with nitration and downregulation of LMO4. Cell Death Discov. 1, Artn 15052 https://doi.org/10.1038/Cddiscovery.2015.52 (2015) Yasuda, M., Kishimoto, S., Amano, M. & Fukushima, S. The involvement of pregnane X receptor-regulated pathways in the antitumor activity of cisplatin. Anticancer Res. 39, 3601–3608. https://doi.org/10.21873/anticanres.13507 (2019) Chaudhuri, A. R. & Nussenzweig, A. The multifaceted roles of PARP1 in DNA repair and chromatin remodelling. Nat. Rev. Mol. Cell Biol. 18, 610–621. https://doi.org/10.1038/nrm.2017.53 (2017) Chaitanya, G. V., Steven, A. J. & Babu, P. P. PARP-1 cleavage fragments: Signatures of cell-death proteases in neurodegeneration. Cell Commun. Signal. 8, Artn 31 https://doi.org/10.1186/1478-811x-8-31 (2010) Specific proteolytic cleavage of poly(adp-ribose) polymerase—An early marker of chemotherapy-induced apoptosis Ju, S. M. et al. Phosphorylation of eIF2 alpha suppresses cisplatin-induced p53 activation and apoptosis by attenuating oxidative stress via ATF4-mediated HO-1 expression in human renal proximal tubular cells. Int. J. Mol. Med. 40, 1957–1964. https://doi.org/10.3892/ijmm.2017.3181 (2017) Brozovic, A. et al. Long-term activation of SAPK/JNK, p38 kinase and Fas-L expression by cisplatin is attenuated in human carcinoma cells that acquired drug resistance. Int. J. Cancer 112, 974–985. https://doi.org/10.1002/ijc.20522 (2004) Lee, D., Lee, S. R., Kang, K. S. & Kim, K. H. Benzyl salicylate from the stems and stem barks of Cornus walteri as a nephroprotective agent against cisplatin-induced apoptotic cell death in LLC-PK1 cells. RSC Adv. 10, 5777–5784. https://doi.org/10.1039/c9ra07009e (2020) Lau, A. H. Apoptosis induced by cisplatin nephrotoxic injury. Kidney Int. 56, 1295–1298. https://doi.org/10.1046/j.1523-1755.1999.00687.x (1999) Ryan, M. J. et al. HK-2: An immortalized proximal tubule epithelial cell line from normal adult human kidney. Kidney Int. 45, 48–57. https://doi.org/10.1038/ki.1994.6 (1994) Hauschke, M. et al. Neutrophil gelatinase-associated lipocalin production negatively correlates with HK-2 cell impairment: Evaluation of NGAL as a marker of toxicity in HK-2 cells. Toxicol. In Vitro 39, 52–57. https://doi.org/10.1016/j.tiv.2016.11.012 (2017) Handl, J. et al. Transient increase in cellular dehydrogenase activity after cadmium treatment precedes enhanced production of reactive oxygen species in human proximal tubular kidney cells. Physiol. Res. 68, 481–490. https://doi.org/10.33549/physiolres.934121 (2019) Capek, J., Hauschke, M., Bruckova, L. & Rousar, T. Comparison of glutathione levels measured using optimized monochlorobimane assay with those from ortho-phthalaldehyde assay in intact cells. J. Pharmacol. Tox. Met. 88, 40–45. https://doi.org/10.1016/j.vascn.2017.06.001 (2017) Download references The financial support was received from the Ministry of Education Youth and Sports of the Czech Republic via projects NANOBIO (Reg Department of Biological and Biochemical Sciences The original online version of this Article was revised: The original version of this Article contained an error in the spelling of the authors Pavlina Majtnerova Jiri Handl and Tomas Rousar which were incorrectly given as Majtnerova Pavlina Download citation DOI: https://doi.org/10.1038/s41598-021-91380-3 Helen Virginia Hoechst passed away April 22 She was married for 30 years to the late Elmer Joseph Hoechst She was the beloved daughter of the late Calvin and Zana (Blackburn) Derrick and was preceded in death by her nine brothers and sisters: Ruth Johnson Lucille Giles and also by her daughter Susan Helen Hoechst Helen was a “giver” who thought of others before herself She would call people who lived alone or had health issues even when she didn’t feel well herself In her final illness she was concerned about her son She was unlikely to complain about anything and was able to “stay in the middle” emotionally She did not ask for much — a real challenge when it came to finding Christmas or birthday gifts for her Helen was always curious about what might be outside — deer She enjoyed watching hummingbirds at the kitchen window as well as the variety of birds that came for some seed She was very organized and did not like clutter— a trait that was not passed on to her children maintaining basically the same routine and schedule daily with certain days allocated for a particular activity cooking something special or grocery shopping Helen lost a significant amount of weight years ago by making changes in her diet and was able to stick with it for the remainder of her life She will always be loved and greatly missed Memorials may be sent to a favorite charity Your name will appear next to the candle you light.Your condolence may need to be approved before it appears on this page It may not appear immediately once submitted Upload attachment (Allowed file types: jpg Δdocument.getElementById( "ak_js_1" ).setAttribute( "value" It looks as though you’ve already said that!' + ' Your condolence may need to be approved before it appears on this page Thank you for leaving a condolence..Your comment has been submitted for moderation Helen V. Hoechst Born May 13, 1927 Departed April 22, 2023 Helen Virginia Hoechst passed away April 22, 2023 at almost 96 years of age. She was married for 30 years to the late Elmer Joseph Hoechst. She was the beloved daughter of the late Calvin and Zana (Blackburn) Derrick and was preceded in death by her nine brothers and sisters:Continue Reading Δdocument.getElementById( "ak_js_2" ).setAttribute( "value" Designed and produced by | doesn’t generally strike people as an excursion destination But it has charming surprises in store for those who give it a chance: a couple of palaces an old town center full of half-timbered houses one of Frankfurt’s oldest churches and the city’s only ferry across the Main River My wife and I headed there on a recent sunny Saturday which is one of the three days of the week the Hoechst market is in operation strolling through the array of vendor stands There has been a market in Hoechst since 1355 and today’s market square was laid out in the 1920s especially at the stands selling coffee and drinks on a Saturday morning We had freshly pressed apple juice and then went out to see the Altstadt Hoechst is full of narrow cobblestone lanes We walked along a couple of these before coming to the highlight of old Hoechst started out as a castle toll station on the Main for the archbishops and electors of Mainz Frankfurters upset with paying the tolls destroyed it in 1396 it was turned into a residential palace by an archbishop at the end of the 16th century but suffered damage in the Thirty Years War the palace served as AFN’s Europe headquarters until 1966 There’s a wonderful view of the Main from the palace gardens and visitors can walk through part of the former moat We had lunch at a restaurant on the palace square then walked down to the Main through two gates of the medieval fortifications We strolled to the pedestrian and bike ferry that connects Hoechst with Schwannheimer Dune The triangle between the rivers is a popular rest and recreation area We watched the ships and the ducks ply the Main for a bit then headed back into town past the Bolongaropalast a Baroque palace built for a pair of snuff manufacturers in the 18th century it was known for its porcelain manufacturing we walked around back and strolled through the church’s small flower and herb garden Justin’s is one of the oldest churches in the area The Baroque high altar and the organ are from the 18th century past the Renaissance old City Hall with its stepped gable roof I wondered why it took so long for us to discover this little gem Location: About 16 miles east of Wiesbaden Friday and Saturday; the Porzellanmuseum is open 11 a.m Metrics details stomata regulate water loss through transpiration for plant growth and survival in response to various environmental stressors; and simple methods to assess stomatal dynamics are needed for physiological studies we report a fluorescence-imaging-based method using fluorescein diacetate tagged with Hoechst 33342 a nuclear staining chemical probe (HoeAc2Fl) for the qualitative assessment of stomatal dynamics the stomatal movement is inferred by simple monitoring of the fluorescence intensity in the nucleus of the stomata Herein, we report a simple and convenient method for the assessment of stomatal closing/opening in Arabidopsis thaliana based on fluorescent live imaging by Hoechst-tagged acetylfluorescein (HoeAc2Fl, Fig. 1a). Our method enables objective assessment of stomatal dynamics by simple monitoring of the fluorescence intensity of HoeAc2Fl in the nucleus of the stomata. (a) Chemical structure of HoeAc2Fl which comprises Hoechst 33342 and fluorescein diacetate moieties (b) Schematic illustration of selective staining of the nucleus in the closed stomata by HoeAc2Fl we proposed that HoeAc2Fl could constitute a useful chemical tool for the assessment of stomatal dynamics by simple monitoring of the intensity of the fluorescence of the nuclei of the guard cells Fluorescent microscopic images of stomata of Col-0 stained by Hoechst 33342 (20 µg/mL (b) in the dark (top) or light (bottom) conditions This result indicates that no transporter is involved in the efflux of HoeAc2Fl from the opened stomata A mechanistic explanation for the selective nuclear localization of HoeAc2Fl is therefore unclear (a) Fluorescent microscopic images of HoeAc2Fl-stained stomata of Col-0 treated with ABA (10 µM) in the light (b) Stomatal aperture of Col-0 treated without or with ABA (10 µM) (c) Relationship between stomatal apertures and fluorescence intensity of the nucleus of HoeAc2Fl-stained guard cells in the mock condition (black circle) in the absence (red cross) or the presence of ABA (blue diamond) (d) Dotted plot of the fluorescence intensity of HoeAc2Fl-stained stomata in the mock condition (black circle) in the absence (black cross) or the presence of ABA (black diamond) (e–g) Fluorescent microscopic images of HoeAc2Fl-stained stomata of Col-0 treated with IAA (10 µM (h) Stomatal aperture of Col-0 treated without or with various chemicals (IAA Significant differences were evaluated by one-way ANOVA/Tukey HSD post hoc test (p < 0.01) (i) Relationship between stomatal apertures and fluorescence intensity of the nucleus of HoeAc2Fl-stained guard cells in the mock condition (black circle) or treated with IAA (green square) (j) Dotted plot of the fluorescence intensity of HoeAc2Fl-stained stomata in the mock condition (black circle) or treated with IAA (green square) Bars represent mean fluorescence intensity (n = 25) HoeAc2Fl is proposed as a tool to easily and quickly assess whether plant stomata are open or closed based on its selectivity for the guard cells of closed stomata The mechanistic basis for this selectivity is unknown the fluorescence was observed only from closed stomata The clear threshold of the fluorescence provides objective criteria for the assessment of stomatal dynamics Instant determination of stomatal dynamics by measuring the fluorescence of HoeAc2Fl with objective analyses is expected to enable high-throughput screening of chemical libraries which may lead to the discovery of novel chemical probes that can improve our understanding of plant responses to changes in their environments and ultimately lead to improved crop production Guard cell signal transduction network: advances in understanding abscisic acid Plant stomata function in innate immunity against bacterial invasion Mechanisms of abscisic acid-mediated control of stomatal aperture Guard cell sensory systems: recent insights on stomatal responses to light The Membrane Transport System of the Guard Cell and Its Integration for Stomatal Dynamics Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins Activation of dimeric ABA receptors elicits guard cell closure Proceedings of the National Academy of Sciences of the United States of America 110 A Rationally Designed Agonist Defines Subfamily IIIA Abscisic Acid Receptors As Critical Targets for Manipulating Transpiration Identification and Characterization of Compounds that Affect Stomatal Movements Insights into the in Vitro and in Vivo SAR of Abscisic Acid – Exploring Unprecedented Variations of the Side Chain via Cross‐Coupling‐Mediated Syntheses European Journal of Organic Chemistry 2018 Potent Analogues of Abscisic Acid – Identifying Cyano‐Cyclopropyl Moieties as Promising Replacements for the Cyclohexenone Headgroup Abscisic acid analogs as chemical probes for dissection of abscisic acid responses in Arabidopsis thaliana Chemical genetic interrogation of natural variation uncovers a molecule that is glycoactivated Use of infrared thermography for monitoring stomatal closure in the field: application to grapevine Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation Arabidopsis HT1 kinase controls stomatal movements in response to CO2 DeepStomata: Facial Recognition Technology for Automated Stomatal Aperture Measurement Ratiometric fluorescence imaging of nuclear pH in living cells using Hoechst-tagged fluorescein Bioorganic & Medicinal Chemistry Letters 27 Programmed induction of endoreduplication by DNA double-strand breaks in Arabidopsis Mithramycin- and 4’-6-diamidino-2-phenylindole (DAPI)-DNA staining for fluorescence microspectrophotometric measurement of DNA in nuclei Journal of Histochemistry & Cytochemistry 29 Enhanced Stomatal Conductance by a Spontaneous Arabidopsis Tetraploid Results from Increased Stomatal Size and Greater Stomatal Aperture Immunosuppressants implicate protein phosphatase regulation of K+ channels in guard cells Proceedings of the National Academy of Sciences of the United States of America 90 K+ channels of stomatal guard cells: bimodal control of the K+ inward-rectifier evoked by auxin Total syntheses of coronatines by exo-selective Diels-Alder reaction and their biological activities on stomatal opening Noncanonical Function of a Small-Molecular Virulence Factor Coronatine against Plant Immunity: An In Vivo Raman Imaging Approach The action of fusicoccin on stomatal guard cells and subsidiary cells Fusicoccin: a Fungal Toxin that opens Stomata Download references P35S::H2B-tdTomato plants of the Col-0 ecotype were kindly provided by Prof Misato Ohtani (Nara Institute of Science and Technology Yasuhiro Ishimaru (Tohoku University) for technical advice This work was supported by a Grant-in-Aid for Scientific Research for MU from MEXT 15H05949 “Resonance Bio” and 18H04546 “Chemistry for Multimolecular Crowding Biosystems”) and JSPS Core-to-Core Program Asian Chemical Biology Initiative (MU) Precursory Research for Embryonic Science and Technology (PREST) Department of Molecular and Chemical Life Sciences Department of Life Science and Applied Chemistry Frontier Research Institute for Materials Science (FRIMS) designed and coordinated the research project Download citation DOI: https://doi.org/10.1038/s41598-020-62239-w went peacefully to Heaven surrounded by her family on Saturday July 27 A funeral service will be held at 11:00 am on Saturday Visitation will be held from 5:00 pm - 7:00 pm on Friday the third of ten children to Albert and Alice Baar She graduated from Dickinson High School in 1971 In 1982 she married Vern Hoechst and assumed the role of mother to Nikki her dream of having her own child came true when Rose was born She worked for Burlington Northern until 1998 and was very involved at First Lutheran Church and going to her 10 grandchildren’s many activities Her greatest joy was spending time with her husband She especially loved cooking meals for the entire family and baking a special cake for each family member’s birthday Wanda was grateful to have shared her life with her husband Nikki (Jason) Van Lishout and Rose (Nolan) Schwarz; her two sons Luke (Anne) Hoechst and Brandon (Loralie) Hoechst Eli and Elin; her siblings and their spouses; and many nieces and nephews MOUNT DORA – Voters in Mount Dora cast their ballots Tuesday for who will lead the city for the next two years ultimately electing Crissy Stile over incumbent Mayor Catherine Hoechst Stile served in the at-large seat from 2018 to 2020 prior to her mayoral bid She received 58.53% of the vote to Hoeschst’s 41.47% [RELATED: 2021 Lake County Voter Guide ] Stile owns Barrel of Books And Games, according to her website which is currently celebrating 10 years of business in Mount Dora is focused on improving business downtown; returning recycling to the city a program that was cut in 2020; building a long-promised recreation center; removing “wasteful pet projects” from the city’s budget; and “smart growth” as Mount Dora’s population grows Hoechst has been serving her second term as mayor She originally served from 2013 to 2015 but lost her bid for reelection to Nick Girone during which time she served on city council Hoechst does not currently have a campaign website When she was reelected to the mayor’s office in 2019 Hoechst promised to focus on the community by working with residents and small businesses in the area; maintaining a balanced budget without diminishing the quality of services taxpayers receive; and building a parking garage in downtown Mount Dora Copyright 2021 by WKMG ClickOrlando - All rights reserved Thomas Mates is a Streaming Executive Producer for News 6 and ClickOrlando.com He also produces the podcast Florida Foodie Thomas is originally from Northeastern Pennsylvania and worked in Portland Oregon before moving to Central Florida in August 2018 He graduated from Temple University with a degree in Journalism in 2010 TV Listings Email Newsletters RSS Feeds Contests and Rules Contact Us / Follow on Social Media Careers at WKMG Closed Captioning / Audio Description Public File Current EEO Report Terms of Use Privacy Policy Do Not Sell My Info FCC Applications EEO Report Disability Assistance Copyright © 2025 ClickOrlando.com is managed by Graham Digital and published by Graham Media Group MOUNT DORA — Mount Dora Mayor Nick Girone knows something about close elections He ran for a city council seat in New Jersey many years ago and was comfortably ahead on election night with just one precinct left to be counted — so comfortably ahead that media and well-wishers crowded him before the final tally was in and he was right: He lost by a meager 33 votes and recalls the sting the next day when friends told him they didn’t even bother to vote because they thought he’d win handily he was running for re-election to the Mount Dora City Council and ended up in a dead heat with challenger Marie Rich the election was decided by drawing a name out of a hat who successfully ran for mayor against Cathy Hoechst four years ago figures to be in another dogfight come Tuesday when Hoechst tries to take the seat back from him Girone believes he’s done enough as mayor in the past four years to earn another two-year term Girone pointed to his work on the Wolf Branch Innovation District an area of east Mount Dora adjacent to the new Wekiva Parkway that city officials hope will attract major commercial and residential development hoping to take advantage of the new road’s easy access to Orlando The city has spent the past four years getting the area ready for development and although it secured a donation of land for a satellite campus of Lake-Sumter State College it has not signed its first major commercial developer at the Innovation District yet Girone wants another term in office to help make that happen “The accomplishment will come when we get that first company signed,” he said “It’s just something I have to do as mayor.” Girone says he is also proud of his efforts to reach out to the largely minority northeast area of the city which he believes has long been overlooked I’d ride around the area to see how things were going and I’d stop in at businesses and asked what I could do and the word they used is inclusion like the downtown area gets all the attention,” he said even the Christmas lights the city strung up in that area around the holidays were hand-me-downs “And that stuck with me that we had to bridge that gap,” he said we can begin to get the two areas together and build one community.” Hoechst is not as pleased with Girone’s performance She believes there is a vacuum in leadership in City Hall and points to high turnover in city staff as evidence She notes that the city has been through several finance directors in recent years and says the Public Works Department has experienced enormous turnover as well Hoechst also expressed concern with the city budget which has nearly doubled in the past three years He notes that the budget appears larger because the city has borrowed money to do a number of public works and infrastructure projects but that the actual operating costs of running the city haven’t changed since Hoechst was in office “Even when you add up all (the public works projects) there’s still $7 million or $8 million that’s additional expenditures in the budget Hoechst believes the issues that plague the city — spending employee turnover and the inability to locate a long-debated city parking garage in downtown — can be traced back to lack of leadership She thinks City Manager Robin Hayes has struggled with some decision-making and she blames Girone for not providing more guidance and accountability who I believe is getting her experience as a city manager on the job with us,” Hoechst said but she should be getting more input and more guidance than she is we wouldn’t be in the place we are with some of these issues.” Hoechst also criticized city leadership for the very public Starry Night fiasco when the city spent a year locked in an expensive legal dispute with a homeowner who commissioned an artist to paint a rendition of Van Gogh’s “Starry Night” masterpiece on her home issued a public apology to the homeowner and paid her legal fees you’ve got to figure out how to make it a win-win for both sides it seemed the city was just trying to win its point and didn’t care to compromise,” she said haven’t put as much energy into locating a parking garage to help downtown businesses as it did opposing a mural on a home She says Girone has simply been too passive “If what it takes is discussing it at every council meeting and getting an update about where we are with it they (the council) talks about it at a council meeting and then they don’t bring it up again for months He says Hayes and her staff have been talking with landowners off and on in hopes of finding a suitable location to buy but they haven’t been able to close a deal that is financially reasonable for the city “I’d be willing to bet you within a year we can have something on paper and that’s whether I’m here as mayor or (Hoechst) is.” As for Hoechst’s criticism of his leadership Girone says by city charter he can’t meddle in the day-to-day workings of city government so he isn’t allowed to tell Hayes how to do her job (Hoechst counters that she’s not talking about micromanaging Hayes but instead providing more assertive guidance) Girone also disputes the notion that Hayes is struggling as a first-time city manager He says she has brought greater accountability and transparency into city government I think to say she’s negligent in any of her duties is just political it’s trying to strike fear into voters and create an issue.” both candidates hope voters will choose them on their leadership qualities and experience Girone is a retired school business administrator serves on the board of the Florida League of Mayors chairs the Lake-Sumter Metropolitan Planning Organization and has chaired or served on the boards of a number of local organizations and events “I enjoy doing for people and helping to get things done for residents,” he said “I want to continue the successes that have been implemented during the past four years and to help to create new ones I am proud to be the mayor of Mount Dora and I believe I earned a chance to continue.” In addition to serving as mayor from 2013-2015 Hoechst is a retired nurse and former manager of Lake Receptions and has been honored on multiple occasions for her volunteer work in the community She was president of the Chamber of Commerce for 10 years and is the current chair of the Lake Cares Food Pantry in addition to serving on various boards over the past 30 years She was the Mount Dora Chamber’s Volunteer of the Year in 1989 and 2015; won a Community Service Award in 2009; and was Lake Magazine Hero in 2009 “I am running for mayor of Mount Dora because I believe we need new leadership,” she said “The citizens of Mount Dora keep sharing similar concerns with me They want clear answers about exactly what progress the community is making to be a better place to live They want elected officials who respect them and are careful in deciding how their hard-earned money is spent Occupation: Retired School Business Administrator; current mayor of the city of Mount Dora Education: BS degree in industrial management from University of Cincinnati Ohio; MA degree in school business administration from Rowan University Education: BS degree in nursing from Florida Southern University; Master’s degree in Healthcare Administration from the Medical College of Virginia.