Supplementary Figure 8: RNAPII inhibition impairs DNA repair and sequence-specific ASOs reduce DDR foci formation at individual DSBs.

(A) Representative images of BJ cells pre-treated with DMSO or DRB for 2 h, irradiated (2Gy) and fixed at the indicated time points (relative to Fig. 6G). Scale bar 5 μm (B) Representative images of neutral comet assay. HeLa cells were pre-treated with DMSO or DRB for 2 h, irradiated (5Gy) and collected at the indicated time points (relative to Fig. 7H, I). Scale bar 20 μm (C) Quantitative analysis of 53BP1 focus intensity, normalized on Mock sample, of the experiments shown in Fig. 7D. Dots represent individual cells. Black bar represents mean. Error bars indicate SEM (data are shown as pool of n = 3 independent experiments, ≥100 cells analysed for each sample). (D) NIH2/4 cells were transfected with the indicated ASOs, irradiated (2Gy) and fixed 1 h post IR. Bar plot shows the percentage of DDR-positive cells (mean of n = 2 independent experiments, >50 cells per sample). (E) Schematic representation of ASOs (A and B in red) preventing the interaction between dilncRNAs (light blue) and DDRNAs (dark blue) originating from Tet sequences in NIH3T3duo cell line. The black arrows indicate the head-to-head configuration of Tet sequences flanking the I-SceI site. (F) Bar plot shows mean fold change normalized to uncut CTL ASO of enrichment relative to input of γH2AX at DAB1 locus at 50, 1000 bp from DSB. Error bars indicate SEM (n = 3 independent experiments). (G) Bar plot shows the percentage of enrichment relative to the input of Mock sample at DAB1 locus at 50, 1000 bp from DSB. Data are shown as one representative of 3 independent experiments. (H) Bar plot shows the percentage of enrichment relative to the input of 53BP1 or Mock at RYR2 locus cut by I-PpoI in HeLa cells. Data are shown as one representative of 2 independent experiments. (I) Bar plot shows the percentage of enrichment relative to the input of 53BP1 or Mock at an unrelated region on chromosome 22 not cut by I-PpoI in HeLa cells. Data are shown as one representative of 2 independent experiments. P value was calculated using two-tailed t-test. ^∗∗∗∗P < 0.0001. ns indicates not significant. Statistical source data are provided in Supplementary Table 4.