Fig. 2.

Temporal changes to BRCA1 and 53BP1 IRIF distributions. (A) RPE1 cells were G0/G1 arrested by serum starvation or asynchronous cell cultures were pulse-labelled with EdU (5 minutes, 20 µM) immediately before irradiation (2 Gy). 30 minutes later, fixed cells stained for EdU in click reactions, were counterstained for BRCA1. Representative projection images constructed from 3D-SIM-acquired Z-series datasets are shown (n≥10 per stage). (B) Cells treated exactly as in A immunostained for 53BP1. (C) Projections and 3D reconstructions of 53BP1 distribution in IRIF (top and bottom two panels, respectively) were assembled from 3D-SIM datasets of cell volumes indicated in B. (D) G1 to S-phase progression is associated with decreased 53BP1 IRIF occupancy. Volumes of isosurface rendered 53BP1 foci from multiple irradiated (harvested 1 hour following 2Gy) RPE1 (n = 32; >300 foci scored per group) and HeLa cells (n = 36; >700 foci scored per group). S-phase position was discriminated as above. EdU-negative cells with a DAPI content <S or G2 were discriminated as G1. Whiskers indicate the 5th to 95th percentile. ***P<0.0001, Mann–Whitney test.