DR‐GFP homologous recombination assay

AsPC‐1 and Panc 08.13 cells were transfected with pDR‐GFP plasmid (a gift from Maria Jasin; Addgene #26475) (Pierce et al, 1999). Cells were selected with puromycin (1.2 µg/ml), and single‐cell clones with stably integrated DR‐GFP plasmid were generated. Genomic DNA was isolated from the single‐cell clones using QIAamp DNA mini kit (Qiagen #51304), and qRT–PCR was performed using DR‐GFP‐CN1 and DR‐GFP‐CN2 primers (Table EV2) to assess the pDR‐GFP integration. The expression of DR‐GFP inserts was normalized to ZNF80 and GPR15 genes (Table EV2). A HeLa‐DR‐GFP clone (Parvin et al, 2011) with a single copy of pDR‐GFP was used as a control to determine the copy number in AsPC‐1 and Panc 08.13 DR‐GFP single‐cell clones.

AsPC‐1 and Panc 08.13 DR‐GFP clones were seeded at optimal density in 24‐ or 12‐well plates and treated with DMSO or 100 nM ETC‐159 for 48 h. The cells were transfected with pCBASceI (a gift from Maria Jasin; Addgene #26477) (Richardson et al, 1998) or pcDNA3.2/V5‐DEST (control) plasmid using Lipofectamine2000 (Thermo Fisher Scientific, #11668500) After 6 h, cells were again treated with DMSO or ETC‐159. After 2 days, cells were harvested by trypsinization, acquired on BD LSRFortessa, and analyzed using FlowJo v10 software for expression of GFP. The percentage of GFP‐positive cells was used as a measure of cells undergoing homologous recombination.