Ionizing radiation-induced foci formation assay

Lig4^−/−, Lig4^−/−:Lin37^−/−, and Lig4^−/−:Trp53bp1−^/− abl pre-B cells were treated or not with 3 μM imatinib for 48 hr. Thereafter, the cells were pulsed with 10 μM EdU (Invitrogen) for 30 min and subjected to 10 Gy irradiation. For the detection of RAD51 foci, irradiated cells were allowed to recover for 4 or 20 hr, at which point they were immobilized on slides pre-coated with CellTak (Corning) and briefly pre-extracted (20 mM HEPES, 50 mM NaCl, 3 mM MgCl₂, 0.3 M sucrose, and 0.2% Triton X-100) on ice for 15 s to remove soluble nuclear proteins. Extracted samples were then fixed (4% paraformaldehyde), permeabilized (0.5% Triton X-100 in PBS), incubated with anti-RAD51 primary antibody (Abcam, ab176458, 1:250). Alternatively, irradiated (10 Gy) cells were allowed to recover for 1 hr prior to fixation without a preceding pre-extraction step, and subsequently incubated with primary antibodies recognizing 53BP1 (Novus Biologicals, NB100-305, 1:1000) or RIF1 (gift from Davide Robbiani [Rockefeller University, New York], 1:5000). In all cases, IRIFs were visualized by incubating samples with Alexa Fluor 555-conjugated secondary antibodies (Invitrogen). Where indicated, click-IT chemistry was performed as per the manufacturer’s instructions. Finally, DNA was counterstained with DAPI (Thermo Fisher Scientific). Immunofluorescence images were captured at 40× magnification on a Lionheart LX automated microscope (BioTek Instruments, Inc). Quantification of IRIF was performed using the Gen5 spot analysis software (BioTek Instruments, Inc).