Extended Data Fig. 4: Disruption of ordered, circular arrangement of DSB-flanking chromatin after depletion of RIF1 or 53BP1.
From: Stabilization of chromatin topology safeguards genome integrity
a, Western blotting of U2OS cells treated with control or two RIF1 siRNAs immunostained for RIF1 and loading marker (tubulin). b, 3D-SIM of GFP–53BP1-MDs in U2OS cells transfected with RIF1 siRNA #2 and treated as in Fig. 1d. c, 3D-SIM of 53BP1-MDs in U2OS cells expressing siRNA-resistant GFP–53BP1-7A mutant and depleted for endogenous 53BP1 (#2 siRNA), exposed to irradiation (1 Gy, 2 h) (top). A schematic depiction of 53BP1-7A where glutamines in 7 SQ/TQ sites are converted to alanines (bottom). d, Distribution of circular with central interchromatin space (IC centre) versus elongated (no IC centre) 53BP1-MDs in U2OS, HeLa Kyoto, RPE1-hTERT and BJ cells (n = 130 per condition) in control or RIF1-depleted cells treated with irradiation (1 Gy, 2 h). e, 3D-SIM of immunostained 53BP1-MDs in U2OS, HeLa Kyoto, RPE1-hTERT and BJ cells after control or RIF1 depletion and irradiation exposure (1 Gy, 2 h). f, A representative 3D view of an ordered, circular arrangement of GFP–53BP1-NDs in wild-type conditions (top) and disordered, elongated shapes after RIF1 depletion (bottom). MIP, maximal intensity projection; 3D opacity view is displayed in three orientations (V1–3) indicated by coloured arrows. All 3D-SIM images in this study were routinely inspected in this way. g, Western blotting of U2OS cells treated with TP53BP1 siRNA and immunostained for 53BP1 and loading marker (NUDC). h, 3D-SIM of γH2AX-MDs in U2OS cells transfected with TP53BP1 siRNA and exposed to irradiation (1 Gy, 2 h). i, 3D-SIM of GFP–53BP1-MD in U2OS cells immunostained for γH2AX and treated as in Fig. 1d. Insets in b, c, h represent magnified single 53BP1-MDs. Scale bars, 5 μm in whole-nucleus images (b, c, h), 200 nm in e, f, i and insets (b, c, h). Experiments were biologically replicated twice with similar results. For detailed image information see Supplementary Table 1. For gel source data see Supplementary Fig. 1.
