Extended Data Fig. 7: Analysis of RIF1 depletion, shieldin localization, and RIF1 recruitment dynamics in the context of DSB-flanking chromatin.
From: Stabilization of chromatin topology safeguards genome integrity
a, b, QIBC of fluorescence intensities associated with γH2AX-MDs (a; n = 1,000 cells per condition) and 53BP1-MDs (b; n = 1,800 cells per condition) in control or RIF1-depleted cells treated with irradiation (1 Gy) as indicated. Box plots: centre line, median; box limits, 25th and 75th centiles; whiskers, minimum and maximum; dots, outliers. ***P = 2.0631 × 10−10, **P = 4.8803 × 10−04, NS P = 0.8651 (a, left to right); ***P = 3.887 × 10−9, NS P = 0.7172 (b, left to right); two-tailed non-parametric Wilcoxon rank-sum test. c, Confocal and STED acquisitions of immunostained 53BP1-MDs in U2OS cells treated with control or RIF1 siRNAs, exposed to irradiation (1 Gy, 2 h) and displayed as single and overlay images. d, Counts of 53BP1-NDs per 53BP1-MD quantified from STED images in c (n = 70 per condition); horizonal bar shows median, P = 0.2711 (left), 0.9566 (right); Cochran–Armitage chi-square test. e, U2OS cells expressing endogenously tagged 53BP1–GFP were treated by laser microirradiation and immunostained for γH2AX and RIF1. Stars indicate times when γH2AX, 53BP1 and RIF1 were first detected at DSBs. f, 3D-SIM of 53BP1-MD and 3×-Flag-SHLD3 in U2OS cells exposed to irradiation (1 Gy, 2h) and immunostained for 53BP1 and Flag-tag (six independent examples are shown). Scale bars, 200 nm (c, f), 20 μm (e). Experiments were biologically replicated twice with similar results. For detailed image information see Supplementary Table 1. For gel source data see Supplementary Fig. 1.
