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. 2020 Apr 14;11:1792. doi: 10.1038/s41467-020-15615-z

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a, b IncuCyte proliferation assay on HCC1569 cells treated with the indicated HDACi. HCC1569 cells used in these experiments contain an inducible H1.0-targeting shRNA (shH1.0_1), which is either not expressed (Quisinostat/Abexinostat) or expressed to prevent H.10 upregulation (Quisinostat/Abexinostat + H1.0 KD). See also Supplementary Fig. 5e, h (independent H1.0-targeting shRNA and a non-targeting shRNA). Values represent mean ± s.e.m. from four biological replicates. Similar results were obtained in three independent experiments. p-value: one-tailed Student’s t-test calculated at the last time point. c Clonogenic assays measuring self-renewal ability of HCC1569 cells. The percentage of wells (n = 96) populated by colonies is shown. Cells contain an inducible H1.0-targeting shRNA (shH1.0_1) which is not expressed (Quisinostat) or expressed to prevent H1.0 upregulation (Quisinostat + H1.0 KD). Cells were treated with 12.5 nM Quisinostat with (H1.0 KD) or without 0.5 µg ml⁻¹ Doxycycline. With DMSO-treated H1.0 KD cells, the percentage of populated wells was 39.6%. The significance of the overall difference among the conditions is indicated (three-way contingency table analysis and two-tailed Fisher’s exact test). d Tumor maintenance assay. Growth kinetics of HCC1569-induced tumors treated with 4 mg kg⁻¹ Quisinostat or vehicle. Injected cells contain an inducible H1.0-targeting shRNA (shH1.0_1) which is either not expressed (Quisinostat) or expressed to prevent H1.0 upregulation (Quisinostat + H1.0 KD). Values represent mean ± s.e.m. from eight tumors. p-value: one-tailed Student’s t-test calculated at the last time point. Arrow: start of treatment. Similar results were obtained in an independent experiment. See also Supplementary Fig. 5k. e Immunodetection of proliferating (Ki67⁺) cells by immunohistochemistry in one of three tumors per condition. Scale bar: 50 µm. f Limiting-dilution transplantation assay into NSG mice using cells from HCC1569-induced tumors treated with the indicated substances. The significance of the overall difference among the conditions is indicated (χ² test). n = 6 tumors. Outgrowths/injections for H1.0 KD: 4/6, 0/6, 0/6 and 0/6 for 10,000, 1000, 100, 10 injected cells, respectively. For the other conditions, see Fig. 2g. Data for all graphs in Source Data file.