Fig. 5
Alt-NHEJ partially contributes to gross genomic instability in BRCA2-depleted cells. a Schematic representation of the EGFP-based alt-NHEJ reporter assay. b BRCA2 depletion leads to an increased alt-NHEJ frequency. Wild-type or Polθ-deficient U2OS alt-NHEJ-EGFP cells were transfected with control siRNA or siRNA against BRCA2. 24 h posttransfection, 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 Polθ partially suppresses gross genomic instability caused by BRCA2 depletion. Wild-type or Polθ-deficient HeLa cells were transfected with control siRNA or siRNA against BRCA2. Forty-eight hours posttransfection, 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 and RAD51 foci and DAPI-stained nuclei are shown. e Quantification of RPA2 and RAD51 foci and nuclear fragmentation. Data represent mean ± SEM of three independent experiments. Over 100 cells were counted in each experiment. f Wild-type, Polθ-, XRCC4-, or Polθ/XRCC4-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 and RAD51 foci and DAPI-stained nuclei are shown. g Quantification of RPA2 and RAD51 foci and nuclear fragmentation. Data represent mean ± SEM of three independent experiments. Over 100 cells were counted in each experiment. h Knockdown/knockout efficiency was confirmed by western blotting. Scale bars, 10 μm
