Fig. 4.

BRCA1-dependent 53BP1 exclusion from IRIF coincides with S-phase DSB repair and 53BP1-associated chromatin displays reduced γH2AX. 48 hours following control (CNTL) or BRCA1 siRNA treatment, HeLa cells were processed exactly as in Fig. 2D. (A) Representative images of two independent experiments in which IRIF (>40 per cell; n≥19) were scored per experimental group. (B) Quantification of experiment shown in A (top). Whiskers indicate the 5th to 95th percentile. ns, not significant (P = 0.8639). ***P<0.0001, Mann–Whitney test. Western blot (bottom) showing BRCA1 protein depletion in CNTL and BRCA1 siRNA-treated cells (lanes 1 and 2, respectively). (C) Cells recovered for 30 minutes following acute CPT treatment (25 nM for 10 minutes) were detergent pre-extracted and fixed before BRCA1 and 53BP1 immunostaining. Representative projection images are presented (n = 10). (D) Irradiated cells (2 Gy) processed as in C were immunostained for CtIP and 53BP1. (E) 72 hours following siRNA treatment, cells were irradiated (2 Gy), recovered (30 minutes), then processed as in B with 53BP1 and γH2AX antisera. Representative projection images are presented (n≥10 in two independent experiments). (F) Projection and 3D-reconstruction images constructed from two-channel 3D-SIM datasets of cell volumes indicated in E.