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. 2018 Sep 12;9:3704. doi: 10.1038/s41467-018-06066-8

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© The Author(s) 2018

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. 2 — Maintenance of ground-state chromatin compaction ensures genome integrity a Design of the experiment. U2OS cells synchronized by double thymidine block were transfected with Control and SET8 siRNA. Cells were released into thymidine-free medium and harvested at the indicated time points. b Cells from a were fixed and stained with γH2A.X antibody and PI followed by flow cytometric analysis. c Bars represent percentage of γH2A.X-positive cells harvested at 15 h post G1/S release. Average ± SD of five independent experiments. ****p < 0.0001 (unpaired t test). n > 20,000 in each experiment. d U2OS cells synchronized as in a were harvested at 15 h post G1/S release and immunoblotted with the indicated antibodies. e U2OS cells synchronized and siRNA transfected as in a were fixed at 15 h post G1/S release and stained with DAPI and γH2A.X antibody. (Bar, 10 µm). f U2OS cells synchronized and siRNA transfected as in a were prepared for single-cell electrophoresis in neutral electrophoresis buffer at 15 h post G1/S release. g Relative comet-tail moments from experiments in f plotted as means ± S.E.M. *p < 0.05 (unpaired t test). n > 55. h U2OS cells expressing Dox-inducible FLAG-HA-tagged histone H4-wild type (H4K20WT) or histone H4 lysine 20 to alanine or arginine (H4K20A/R) mutant were synchronized with double thymidine block. Cells were fixed at 15 h post G1/S release and stained with γH2A.X antibody and PI followed by flow cytometric analysis. i Bars represent percentage of γH2A.X-positive cells from h. Average ± SD of three independent experiments. *p < 0.05, **p < 0.01 (ANOVA). n > 20,000 in each experiment. j U2OS cells synchronized and siRNA transfected as in a were mock and sucrose treated (125 mM) at 12 h post G1/S release and were fixed at 15 h post G1/S release. Bars represent percentage of γH2A.X-positive cells in the indicated samples (average ± SD of three independent experiments). ****p < 0.0001 (unpaired t test). n > 20,000 in each experiment. k U2OS cells were transfected with mCherry-tagged human RNF2 (RING1b) and were double thymidine synchronized, siRNA transfected and fixed as in (2e) and stained with DAPI and γH2A.X antibody. (Bar, 10 µm)