Extended Data Fig. 6: Live-SIM imaging of 53BP1-MDs with the underlying chromatin and after RIF1 depletion.
From: Stabilization of chromatin topology safeguards genome integrity
a, 3D-SIM of immunostained γH2AX-MDs in control or 53BP1-depleted U2OS cells treated with irradiation (1 Gy) for the indicated times. b, Western blotting of U2OS cells expressing GFP–53BP1 and H2B–Halo-Tag immunostained for 53BP1, GFP, H2B, Halo-Tag and loading marker (MCMBP). c, Live-SIM depicting an evolving GFP–53BP1-MD at a single H2B–HaloTag-labelled chromatin locus after induction of DSBs by NCS (10 ng ml–1) for the indicated time-points. Insets are magnified 53BP1-MDs. Intensity line profiles of the two fluorophores (along the white lines in the insets) show colocalization of underlying chromatin with the 53BP1-MD. Fluorescence intensities were normalized to the maximum value of each profile. d, Additional examples of live-SIM of cells treated as in Fig. 2c. Image galleries for seven fields from four independent acquisitions are displayed. Manual classification of transition stages is colour-coded. Experiments in a–c were biologically replicated twice with similar results. Scale bars, 200 nm in a, d, and insets in c; 1 μm in large fields in c. For detailed image information see Supplementary Table 1. For gel source data see Supplementary Fig. 1.
