Axiovision camera and software

The γ-H2AX, 53BP1, RAD51, and BRCA1 (Millipore, Billerica, MA, USA; Santa Cruz Biotechnology, Santa Cruz Biotechnology, Santa Cruz, CA, USA and Novus Biologics, Littleton, CO, USA, respectively) IR-induced foci assay was performed as previously described.^{43 (link)} For γ-H2AX and 53BP1 foci, cells were treated with 2 Gy and collected at 1, 4, 8, and 24 h. Cells were collected at 4, 8, and 24 h post-12 Gy for assessment of BRCA1 foci. RAD51 foci were assessed 8 h post-IR, as previously described.^{43 (link)} Cells were plated on coverslips, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton-X 100 (Sigma-Aldrich), blocked in 10% FBS, and incubated with primary antibodies (1 : 300) for 1 h at room temperature. The coverslips were washed, blocked with 10% FBS, and incubated with a AlexaFluor-488 secondary antibody (1 : 400, Invitrogen) for 45 min at room temperature. Coverslips were washed a final time and mounted on slides using ProlongGold anti-fade reagent containing DAPI (Applied Biosystems, Grand Island, NY, USA). Foci were imaged using an Olympus fluorescent microscope equipped with an AxioVision camera and software. Cells were scored as positive if they contained four or more foci/nuclei and results are presented as percent positive cells (% Pos).