Fig. 3: ZFP281 depletion leads to aberrant R-loop accumulation.
From: ZFP281-BRCA2 prevents R-loop accumulation during DNA replication
a Representative immunofluorescence images with the S9.6 antibody showing the levels of R-loop in WT, ZFP281 KO-1, and ZFP281 KO-2 ES cells. DNA was counterstained using DAPI. Three independent experiments show similar results. b Fluorescence intensities of R-loop loci in WT (n = 48), ZFP281 KO-1 (n = 66) and ZFP281 KO-2 (n = 53) ES cells. Three independent experiments show similar results. Error bars represent 95% confidence intervals. Two-tailed, unpaired Student’s t tests were performed. (WT vs. ZFP281 KO-1, p < 0.0001; WT vs. ZFP281 KO-2, p < 0.0001.) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. = not significant. c Immunofluorescence imaging of H3S10P and H3T3P in WT, ZFP281 KO, and ZFP281 KO with RNase H1 overexpression ES cells. White arrows showing the interphase cells positive for H3S10P. d Percentage of WT (total n = 369), ZFP281 KO (total n = 380) and ZFP281 KO with RNase H1 overexpression (total n = 290) ES cells in mitosis and interphase. Mean ± SEM from three independent experiments. Two-tailed, unpaired Student’s t tests were performed. (Mitosis, WT vs. ZFP281 KO, p = 0.0574; ZFP281 KO vs. ZFP281 KO + RNase H1, p = 0.5526. Interphase, WT vs. ZFP281 KO, p = 0.0020; ZFP281 KO vs. ZFP281 KO + RNase H1, p = 0.0016.) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. = not significant. e Western blot analysis (upper panel) showing the levels of chromatin-bound γH2A.X and PCNA in WT, ZFP281 KO, and ZFP281 KO with RNase H1 overexpression ES cells after release from APH treatment for 0, 0.5 and 1 h respectively. Histone H3 was used as a loading control. Gray values (middle and lower panels) of γH2A.X and PCNA protein bands were determined by Image J and normalized to the loading control. Mean ± SEM from three independent experiments. Multiple t tests were performed. (γH2A.X in WT and ZFP281 KO, DMSO, p < 0.0001; R0, p = 0.0005; R0.5, p < 0.0001; R1, p = 0.0035. γH2A.X in ZFP281 KO and ZFP281 KO + RNase H1, DMSO, p < 0.0001; R0, p = 0.0003; R0.5, p < 0.0001; R1, p = 0.0001. PCNA in WT and ZFP281 KO, DMSO, p < 0.0001; R0, p = 0.0002; R0.5, p < 0.0001; R1, p < 0.0001. PCNA in ZFP281 KO and ZFP281 KO + RNase H1, DMSO, p < 0.0001; R0, p < 0.0001; R0.5, p < 0.0001; R1, p < 0.0001.) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. = not significant.
