Figure 3. DDX5‐ and BRCA2‐depleted cells accumulate DNA‐RNA hybrids at DSBs sites.

1. Left: Representative images of in situ PLA between S9.6 and γH2AX antibodies in U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5), or control cells (siC). When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24 h before or treated with cordycepin (Cordy) for 2 h at 37°C before fixation. Single antibody controls from non‐irradiated siC cells are shown. Scale bar indicates 10 µm. Nuclei as defined by auto threshold plugin on the DAPI image (ImageJ) are outlined in yellow. Right: Quantification of PLA spots per nucleus in each condition as indicated. At least 500 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples, we applied a Kruskal–Wallis test followed by Dunn’s multiple comparison test and the P‐values show significant differences. The red line in the plot indicates the median, and each symbol represents a single PLA spot. See also Fig EV4A.
2. DRIP‐qPCR signal values at RBMXL1 and HIST1H2BG loci in U2OS DIvA cells transfected with the indicated siRNAs and treated in vitro with RNase H1 (RH) pre‐immunoprecipitation where indicated. The experiment was performed in both untreated cells (−OHT) and after tamoxifen addition (+OHT). The data represent the mean ± SEM from at least four independent experiments. The statistical significance of the difference was calculated with unpaired one‐tailed t‐test, and the P‐values show the significant difference. See also Fig EV4B and C.