Rad51 foci assay

About 50,000 cells were plated on each Poly-lysine-coated coverslip in a 24-well plate. One day after plating, treatment was added to the cells. Twenty-four hours after treatment, cells were pre-extracted on ice for 3 min with ice-cold 0.5% Triton X-100 followed by fixation with 3% PFA for 15 min at room temperature. After PBS washes, cells were permeabilized with ice-cold 0.5% Triton X-100 for 5 min and blocked in 3% BSA made in PBS for 30 min at room temperature. EdU was labeled with Alexa-Fluor Azide using Click Reaction by following the manufacturer’s instructions (Fisher Scientific, 502108139). Cells were washed once with 3% BSA in PBS, thrice with 0.2% PBS-Tween, and once with PBS. Cells were fixed again with 3% PFA for 10 min, followed by PBS washes. Samples were incubated with 1:500 dilution of rabbit anti-Rad51 antibody (Cosmo Bio, BAM-70-001-EX) for 1 h at room temperature, followed by washes with 0.2% PBS-Tween. Samples were incubated with 1:1000 dilution of Alexa-Fluor 555 conjugated anti-rabbit secondary (Thermo, A32732) for 1 h at room temperature. Coverslips were mounted using Prolong Diamond Antifade mounting media with DAPI (Thermo, {"type":"entrez-protein","attrs":{"text":"P36962","term_id":"547832","term_text":"P36962"}}P36962). All images were acquired on a Nikon Ti-2 60x objective. For cells, z-stacks were taken at every 0.2 micron. The stacks were subjected to deconvolution followed by extended depth focus analysis to obtain the 3D projections. ROI for each nucleus was defined using a magic wand tool in the DAPI channel. The number of Rad51 foci for each ROI was quantified using the Find Maxima function in ImageJ. All analysis was blinded.