Fig. 4

SSA is not responsible for gross genomic instability in BRCA2-depleted cells. a Schematic representation of the EGFP-based SSA reporter assay. b BRCA2 depletion leads to an increased SSA frequency. Wild-type, RAD52-, or ERCC1-deficient U2OS SSA-EGFP cells were transfected with control siRNA or siRNA against BRCA2. Twenty-four hours post transfection, cells were electroporated with pCBASce plasmid. After 48 h, the percentage of GFP-positive cells was determined by FACS. Results represent mean ± SEM of three independent experiments. c Knockdown/knockout efficiency was confirmed by western blotting. d Loss of RAD52 or ERCC1 was unable to reverse gross genomic instability in BRCA2-depleted cells. Wild-type, RAD52-, or ERCC1-deficient HeLa cells were transfected with control siRNA or siRNA against BRCA2. Forty-eight hours post transfection, cells were exposed to 10 Gy IR and then allowed to recover for 2 or 24 h before being processed for immunofluorescence using antibodies against RPA2 and RAD51. Representative RPA2/RAD51 foci and DAPI-stained nuclei are shown. e Quantification of RPA2/RAD51 foci and nuclear fragmentation. Data represent mean ± SEM of three independent experiments. Over 100 cells were counted in each experiment. Scale bar, 10 μm