Fig. 3

BRCA2 deficiency triggers spontaneous DNA damage and G1 arrest. a BRCA2 ^∆Ex3-4/− cells were immunostained for γH2AX. DNA was counterstained with DAPI. Representative images (left) and quantification of γH2AX mean nuclear intensity (right) are shown with analysis by a two-tailed Mann–Whitney test. Median γH2AX intensity, red bars. Scale bars 10 µm. b Cell cycle analysis demonstrates an increase in the G1 fraction upon BRCA2 deficiency. Representative plots displaying results from the same amount of cells for both samples are shown on the left. An unpaired two-tailed t-test was used for the analysis. n ≥ 3. c Western blot showing p53 loss. Cells were harvested 2 h after 10 Gy irradiation (IR). Two independent BRCA2 ^fl/− p53 ^−/− clones were analyzed. d Western blots of nuclear extracts prepared from the indicated cells. e Cell cycle analysis. An unpaired two-tailed t-test was used for the analysis. n ≥ 3. f Cellular senescence, as indicated by SA β-gal staining. An unpaired two-tailed t-test was used for the analysis. n ≥ 3. g p53 loss leads to a partial increase in clonogenic survival of BRCA2-deficient cells. Left: plating efficiency, as analyzed by an unpaired two-tailed t-test. n ≥ 3. Right: images of representative plates. Arrows highlight typical PCR-validated BRCA2 ^∆Ex3-4/− p53 ^−/− colonies. h Apoptosis analysis using Annexin V staining. Analysis is by an unpaired two-tailed t-test. n ≥ 3. Error bars s.d. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001