Rabbit anti 53bp1

P133 cells were fixed in 3.7% formaldehyde/PBS for 10 min, permeabilized with 0.5% Triton/PBS, and blocked with 10% FCS/PBS. The primary antibodies used were mouse anti-γH2AX (Upstate Biotechnology) and rabbit anti-53BP1 (Bethyl Laboratories). Appropriate Cy3- and Cy5-conjugated secondary antibodies were added (Jackson ImmunoResearch Laboratories). Images were taken with a Bio-Rad confocal microscope, and analysis was performed using ImageJ. Colocalization of both signals was considered as a DSB foci. Foci analysis was performed from at least 45 nuclei for each condition.

For immunofluorescence, U2OS cells were grown on cover-slips, the cells were incubated with DMSO or Spiro (40 μM) for 24 h and then mock or Phleomycin (10 μg/ml)-treated for 1 h. For recovery the cells were grown for 2, 6 or 24 h in the presence of DMSO or Spiro (40 μM). In the case of U2OS lacO/ISceI/tet19 cells (U2OS 19), the cells were co-transfected with the plasmid vectors mCherryLacI and HA-ISceI or empty vector and were grown in medium containing Spiro or DMSO for 24 h. Subsequently, the cells were fixed with 4% formaldehyde and permeabilized with 0.5% Triton X 100. Antibodies used are rabbit anti-53BP1 (Bethyl Laboratories), mouse anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Merck Millipore), rabbit anti-RAD51 PC130 (Calbiochem), rabbit anti-BRCA1 sc642 (Santa Cruz), mouse anti-CtIP clone 14–1 (Active Motif) and mouse anti-RPA32 NB600–565 (Novus). For western blots, U2OS cells were lysed in RIPA buffer, the protein content was quantified by Bradford and the samples were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Antibodies used are rabbit anti-phospho-Histone H2A.X (Ser139) ab2893 (Abcam), rabbit anti-H2A.X ab11175 (abcam), rabbit anti-phospho-RPA32 S4/8 A300–245 (Bethyl), mouse anti-RPA32 NB600–565 (Novus) and mouse anti-alpha-tubulin T9026 (Sigma).

The following primary antibodies were used: mouse anti-HA (clone 16B12, 1:1,000, #901533, Biolegend), mouse anti-γ-Tubulin (clone 6H3.1, 1:1,000, #5886, Cell Signalling), rabbit anti-GFP (1:1,000, #2555, Cell Signalling), rabbit anti-53BP1 (1:800, #A300-272A, Bethyl laboratories), rabbit anti-RAD51 (1:1,000, #70-012, Bioacademia), rat anti-BrdU (CldU) (clone [BU1/75 (ICR1)], 1:250, #ab6326, Abcam), mouse anti-BrdU (IdU) (1:40, #347580, BD Biosciences), rabbit anti-CtIP (1:500, #A300-488A, Bethyl Laboratories), mouse anti-Biotin (1:200, #200-002-211, Jackson Immuno Research), rabbit anti-MRE11 (1:500, a kind gift from Arnab Ray Chaudhuri^{19 (link)}).