HN30 and HN31 cell lines with pDRGFP for HR assay (Addgene, Plasmid #26475, RRID:Addgene_26475) or EJ5GFP for NHEJ assay (Addgene, Plasmid 44026, RRID:Addgene_44026) were generated as previously described (19 (link)). The HN30 and HN31 cell lines were transfected with 2 μg mCherry (Addgene, Plasmid 41583, RRID:Addgene_41583) as a negative control, or with both 2 μg mCherry and 6 μg pCBASceI (Addgene, Plasmid 26477, RRID:Addgene_26477). The cells were incubated overnight in 1 mL of media containing DMSO for the controls, 50 μmol/L defactinib, or 50 μmol/L ATM inhibitor (KU-55933, Selleckchem) for HN30 and 10 mmol/L ATM inhibitor (KU-55933, Selleckchem) for HN31 as the positive controls. The media were replaced with 2 mL of media the following day with the same concentrations of drug added for 24 hours. The cells were then trypsinized, centrifuged into a pellet at 1,200 rpm for 5 minutes, and washed with PBS before being resuspended in 1 mL of FACS buffer containing PBS, 0.1% BSA (Sigma) and 0.1% NaA (Sigma). Flow cytometry was run using the BD Accuri C6 Plus flow cytometer (BD Biosciences) to detect GFP and RFP, with cells positive for mCherry and GFP being gated as positive (19 (link)). The data were plotted with GraphPad Prism, RRID:SCR_002798.