Fig. 6 | Nature Communications

Fig. 6

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

Fig. 6The alternative text for this image may have been generated using AI.

Artemis acts as a nuclease to promote alt-NHEJ. a ERCC1 is not required for alt-NHEJ. Wild-type or ERCC1-deficient 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 Artemis loss reduces the frequency of alt-NHEJ. Wild-type or Artemis-deficient 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. c Knockdown/knockout efficiency was confirmed by western blotting. d The nuclease activity of Artemis is indispensable for its role in promoting alt-NHEJ. Artemis-deficient U2OS alt-NHEJ-EGFP cells stably expressing wild-type Artemis or the nuclease-inactivating mutant (H35AD37N) 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. e Artemis loss suppresses gross genomic instability in BRCA2-depleted cells. Wild-type or Artemis-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. Representative RPA2/RAD51 foci and DAPI-stained nuclei are shown. f Quantification of RPA2/RAD51 foci and nuclear fragmentation. Data represent mean ± SEM of three independent experiments. Over 100 cells were counted in each experiment. g The nuclease activity of Artemis is indispensable for its role in promoting gross genomic instability in BRCA2-deficient cells. Artemis-deficient HeLa cells stably expressing wild-type Artemis or the nuclease-inactivating mutant (H35AD37N) were transfected with indicated siRNAs, exposed to 10 Gy IR, and then allowed to recover for 24 h before being processed for immunofluorescence. Representative RPA2/RAD51 foci and DAPI-stained nuclei are shown. h Quantification of RPA2/RAD51 foci and nuclear fragmentation. Data represent mean ± SEM of three independent experiments. Over 100 cells were counted in each experiment. i The expression of wild-type Artemis or its H35AD37N mutant was confirmed by western blotting. Scale bars, 10 μm

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