Double-Strand Break Repair Assay

The NHEJ and HR reporter plasmids, pCBASceI (I-SceI expressing plasmid), and pDsRed2-N1 were kindly provided by Dr Vera Gorbunova (University of Rochester, NY). The experiment was done as previously described (24 (link)). To generate reporter cell lines, the linearized NHEJ or HR reporter cassettes (0.5 μg) were transfected into ParC5 cells using Amaxa Nucleofector 2B (Basic Epithelial Cells Kit, Program T-020, Lonza, Walkersville, MD). Post 24 h transfection, cells were selected with 100 μg/ml G418 for 7–10 days to generate stable ParC5-NHEJ and ParC5-HR reporter lines. For the DSB repair assay, the ParC5 reporter lines were pretreated with DMSO, 50 nM dasatinib, or 10 μM imatinib for 24 h prior to cotransfection with 5 μg I-SceI to induce DSBs and 0.1 μg pDsRed2-N1 to normalize for the differences in transfection efficiency. Cells were treated with DMSO, 50 nM dasatinib, or 10 μM imatinib and allowed to repair for 72 h. Cells were analyzed using a Gallios 561 flow cytometer (Beckman Coulter, Indianapolis, IN) and Kaluza software (Beckman Coulter). The repair efficiency was calculated by the ratio of GFP+ cells to DsRed+ cells.