Extended Data Fig. 9: Disruption of ordered, circular arrangement of DSB-flanking chromatin after cohesin depletion.
From: Stabilization of chromatin topology safeguards genome integrity
a, Western blotting of HCT116-RAD21–mAID–mClover cells treated with auxin (aux) as indicated and immunostained for RAD21 and loading marker (NUDC). b, Widefield images of HCT116-RAD21–mAID–mClover cells, either untreated or treated with auxin for 6 h to induce RAD21 degradation. c, QUANTEX analysis of mean breadth of 53BP1-MDs in cells treated as in Fig. 3c, d (n = 110). Box plots: centre line, median; box limits, 25th and 75th centiles; whiskers, minimum and maximum; dots, outliers. ****P = 3.8495 × 10−17 for MCM+, ****P = 7.636 × 10−16 for MCM–; two-tailed non-parametric Wilcoxon rank-sum test. d, Western blotting of U2OS cells treated with control or RAD21 siRNA, immunostained for RAD21 and loading marker (tubulin). e, Western blotting of U2OS cells treated with control or SMC1 siRNA, immunostained for SMC1 and loading marker (MCMBP). f–h, 3D-SIM of GFP–53BP1-MDs in U2OS cells transfected with RAD21 siRNA (f), SMC1 siRNA #1 (g), or SMC1 siRNA #2 (h) and exposed to irradiation (1 Gy, 2 h). i, Western blotting of U2OS cells treated with the indicated siRNAs and immunostained for γH2AX; total protein stain is loading control. j, Western blotting of U2OS cells treated with indicated siRNAs and immunostained for 53BP1 and loading marker (MCM7). k, l, 3D-SIM of GFP–53BP1-MDs in U2OS cells treated with 10 μM DNA-PK inhibitor (k) or RBBP8 (also known as CtIP) siRNA (l) and exposed to irradiation (1 Gy, 2 h). m, Western blotting of U2OS cells treated with control or RBBP8 siRNA, immunostained for CtIP and loading marker (NUDC). Insets in (f–h, k, l) are magnified 53BP1-MDs. Scale bars, 5 μm in whole nuclei (f–h, k, l), 200 nm in insets (f–h, k, l), 20 μm in b. Experiments were biologically replicated twice with similar results. For detailed image information see Supplementary Table 1. For gel source data see Supplementary Fig. 1.
