MMEJ Efficiency Quantification in PARP Inhibition

In the reporter assay, 2.5 × 10⁵ cells were transfected with siControl and siRB for 3 days. Cells were then treated with DMSO, 1 μM or 10 μM olaparib for 1 h in both siControl- and siRB-treated cells. Cells were then transfected with the EJ2GFP (Addgene, Watertown, MA, USA) to quantify MMEJ efficiency. Furthermore, 1 μg EJ2GFP plasmids was transfected along with 1 μg pCBASceI (Addgene, Watertown, MA, USA) to cells with Fugene6 (Promega, Madison, WI, USA). The ratio of Fugene6 to the transfected plasmid was 3:1. In another round of transfection, the same numbers of cells transfected with 2 μg red fluorescent protein (RFP) expression plasmid pCAG-DsRed (Addgene, Watertown, MA, USA) were used to normalize the transfection efficiency [27 (link)]. Cells were then incubated for 72 h after transfection and collected for flow cytometry analysis.