Fig. 3.

Cells with DNA damage display reduced chromatin mobility. (A) DNA damage induction by BLM was verified in cells expressing mCherry–53BP1_ct. (B) Chromatin diffusion in vehicle- and BLM-treated cells. Quantitative data in A,B show the mean±s.e.m; *P<0.05 (unpaired Student's t-test, n = 20). Representative MSD traces are shown. (C) Schematic of the approach used to measure chromatin diffusion in the context of a single DSB. The DSBs were induced and visualized by coexpressing TetR–mCherry and GR–ISceI in U2OS cells with an integrated Tet array flanking an ISceI recognition site (arrowhead). Confocal images of GFP and mCherry fluorescence are shown (upper panels). Nuclear translocation of GR–ISceI upon triamcinolone acetonide (TA) treatment was confirmed by imaging an RFP tag fused to GR–ISceI, in the absence of triamcinolone acetonide (−TA) or 30 min after triamcinolone acetonide treatment (+TA) (lower panels). Note that the weak RFP signals did not interfere with foci localization. (D) Immunostaining for γH2AX (upper panel) and imaging of PCNA–GFP (lower panel) confirms DSB induction after triamcinolone-acetonide-mediated nuclear translocation of GR–ISceI. Tet arrays are indicated with arrowheads. (E) Chromatin diffusion in cells where triamcinolone acetonide was omitted (uncut) and after DNA cleavage by ISceI (cut). The schematics indicate the position of the PAGFP–H2A spots, either overlapping the cleavage site or not overlapping. (F) Chromatin diffusion after exposure to ionizing radiation (IR). Timecourse measurements during recovery were grouped in 10-minute bins. The data represent the mean±s.e.m. (five experiments); *P<0.05 (unpaired Student's t-test). The induction of DSB after ionizing radiation was confirmed in cells expressing GFP-tagged 53BP1 (right panels). Scale bars: 10 µm.