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. 2020 Aug 14;11:4083. doi: 10.1038/s41467-020-17503-y

Fig. 8. BRD4 loss causes TRCs and replication fork stalling in S-phase.

Fig. 8

a IF images and quantification of γH2AX and S9.6 intensities per nucleus and EdU incorporation in HeLa cells following treatment with ARV-825 (n = 3 separate experiments). γH2AX foci intensity data shown as mean ± SEM. Box-whisker plots for S9.6 intensity drawn as in Fig. 1b. Significance assessed using ANOVA followed by Tukey’s test (γH2AXDMSO EdU (−) vs ARV-825 EdU (+) ****Adjusted P < 0.0001; γH2AXDMSO EdU (+) vs ARV-825 EdU (+) ***Adjusted P = 0.0003; γH2AXARV-825 EdU (−) vs ARV-825 EdU (+) ****Adjusted P < 0.0001; S9.6DMSO EdU (−) vs ARV-825 EdU (−) ****Adjusted P < 0.0001; S9.6DMSO EdU (+) vs ARV-825 EdU (+) ****Adjusted P < 0.0001). Scale bar = 5 μm. b IF images and quantification of pRPA2 ser33 (pRPA), γH2AX, and native BrdU (ssDNA) immunofluorescence in cells treated with ARV-825 shown as mean ± SEM (n = 3 separate experiments). Significance assessed using two-tailed unpaired t test (*P < 0.05). Scale bar = 2.5 μm. c Quantification of the percentage of EdU-positive cells following treatment with DMSO (black), ARV-825 (blue), or JQ1 (red), shown as mean ± SEM (n = 3 separate experiments). Significance assessed using ANOVA followed by Tukey’s test (DMSO vs ARV-825 ****Adjusted P value < 0.0001; DMSO vs JQ1 ****Adjusted P value < 0.0001). d Experimental scheme for DNA fiber combing experiments. Fluorescent images of DNA fiber combing following treatment with either DMSO, ARV-825, or JQ1. Representative examples of normal replication, fork stalling, and stalling re-start, respectively, are shown. e Quantification of fork speed performed following treatment with DMSO (black), ARV-825 (blue), or JQ1 (red). Mean ± SEM from n = 3 separate experiments is shown. Significance assessed using ANOVA followed by Tukey’s test (DMSO vs ARV-825 ****Adjusted P value < 0.0001; DMSO vs JQ1 ****Adjusted P value < 0.0001). Source data are provided as a Source data file.