Supplementary Figure 7: IR-induced DDR foci are sensitive to RNAPII inhibition in normal human fibroblasts.

(A,B) Accumulation of 53BP1, but not γH2AX, to an AsiSI-induced DSB is impaired in AsiSI-ER BJ-5Ta cells treated with DRB at a genic site. The bar plots show the percentage of ChIP enrichment relative to the input of γH2AX (A) and 53BP1 (B) associated with genomic DNA. Data are shown as one representative of 3 independent experiments. (C) Fold change relative to uninduced cells of γH2AX and 53BP1 enrichment at a genic site (relative to A,B). Values are shown as mean. Error bars indicate SEM (n = 3 independent experiments). (D,E) Same analyses as in A,B at an AsiSI-ER intergenic site. Data are shown as one representative of 3 independent experiments. (F) Fold change relative to uninduced cells of γH2AX and 53BP1 enrichment at an intergenic site (relative to D,E). Values are shown as mean. Error bars indicate SEM (n = 3 independent experiments). (G–J) Foci of RIF1 (G), pKAP1 (H), RNF168 (I) and ubiquitinylated proteins detected by FK2 antibody (J) are reduced in irradiated normal human fibroblasts (BJ) treated with DRB for 2 h at RT before IR. The panel shows representative images. Scale bar 5 μm. The bar plot shows the percentage of positive cells (cells with >10 foci for RIF1, RNF168 and FK2 and >3 foci for pKAP1 were considered positive). Error bars indicate SEM (n = 3 independent experiments, >150 cells per sample). P value was calculated using two-tailed t-test. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗∗P < 0.0001. ns indicates not significant. Statistical source data are provided in Supplementary Table 4.