Fig. 7 | Nature Communications

Fig. 7

From: BRCA2 antagonizes classical and alternative nonhomologous end-joining to prevent gross genomic instability

Fig. 7The alternative text for this image may have been generated using AI.

Artemis functions epistatically with 53BP1 and RIF1 to promote alt-NHEJ. a 53BP1 or RIF1 depletion reduces the frequency of alt-NHEJ in both wild-type and BRCA2-depleted cells. 53BP1-, RIF1- or PTIP-depleted U2OS alt-NHEJ-EGFP cells were transfected with indicated siRNAs and then 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. b Knockdown efficiency was confirmed by Western blotting. The Asterisk indicates a nonspecific band. c Artemis functions epistatically with 53BP1 and RIF1 to promote alt-NHEJ. Wild-type or Artemis-deficient U2OS alt-NHEJ-EGFP cells infected with the indicated lentiviral shRNAs 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. d Loss of 53BP1 or RIF1 suppresses gross genomic instability in BRCA2-depleted cells. Wild-type, 53BP1-, PTIP-, or RIF1-deficient HeLa cells were transfected with indicated siRNAs, 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/RAD51 foci and nuclear fragmentation. Data represent mean ± SEM of three independent experiments. Over 100 cells were counted in each experiment. f RIF1 is required for the retention of Artemis at laser-induced DSBs. 53BP1- or RIF1-depleted HeLa cells were transfected with indicated siRNAs. Forty-eight hours later, cells were transfected with GFP-Artemis and were then micro-irradiated and monitored by live cell imaging. Representative images taken at the indicated times after laser microirradiation are shown. g The fluorescence intensity at the microirradiated site was quantified. Data represent mean ± SEM from at least 20 cells in three independent experiments. Scale bars, 10 μm

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