Immunofluorescence experiments were performed on cells grown on 12-mm coverslips for 48 h after seeding. Cells were treated with 200 μg/ml phleomycin (Sigma-Aldrich) for 2 h, medium was replaced, and cells were further incubated at 37°C for the indicated times. Cells were washed with PBS or CSK buffer (10 mM 1,4-piperazinediethanesulfonic acid, pH 6.8, 100 mM NaCl, 3 mM MgCl2, 300 mM glycerol) supplemented with 0.7% IGEPAL CA630 and 0.1 mg/ml RNase A (Thermo Fisher Scientific) for 3 min at room temperature (Britton et al., 2013 (link)). After two washes with ice-cold PBS, cells were cross-linked for 15 min with 4% paraformaldehyde at room temperature. Coverslips were blocked for 1 h at room temperature in blocking buffer (5% BSA in PBS). XRCC5 antibody was diluted 1:500 in blocking buffer and incubated for 2 h at room temperature. Anti-rabbit secondary antibody Alexa Fluor 488 (Thermo Fisher Scientific) was then added for 1 h at room temperature. Coverslips were counterstained with Hoechst 33342 (Thermo Fisher Scientific) and then mounted onto glass slides using ProLong Gold Antifade reagent (Thermo Fisher Scientific). Images were acquired using a Zeiss LSM 510 microscope.