Pdr gfp plasmid

To establish stable cell lines harboring the DR-GFP gene cassette, the pDR-GFP plasmid (Addgene, #26475) was transfected into PC-3 or A549 cells to get puromycin-resistant clones. Next, 5 μg of pCBASce1 (Addgene, #26477) and 1.5 μg of pDsRed-N1 (Clontech, Mountain View, CA, USA) were co-transfected into the stable cell lines, and cells were treated with drugs or vehicle control for 12 h.

To measure the capacity of cells to repair DSB by HR, LN229 cells were stably transfected with a pDRGFP plasmid (Addgene). The plasmid bears two non-functional GFP genes. One truncated, the other containing a recognition site for I-SceI endonuclease. Upon transient transfection with I-SceI expressing plasmid (Addgene), the endonuclease cleaves the modified GFP gene leading to a DSB. If this DSB is getting repaired by HR, using the sequence of the truncated GFP gene, a functional GFP protein is generated, which can be quantified by flow cytometry. Cells were analyzed 72 h after transfection with 1 μg pCβASceI using the transfection kit Effectene (Quiagen). During transfection, the cells were incubated in the presence of ART (24 h, 48 h, 72 h). The DNA-PK inhibitor KU0060648 (Selleckchem) was used for comparison at a final concentration of 1 μM. We expected that the inhibition of DNA-PK leads to a moderate increase in HR activity due to the lack of competition by NHEJ for the repair of DSB (Shrivastav, 2008). Cells were trypsinized and washed with PBS and measured by flow cytometry using FACS Canto II (BD Biosciences). Data were analysed with BD FACSDiva™ software.

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.