shRNA transfection

U2OS, HCT116, or PEO-1 cells were seeded in a six-well plate with 2 ml of complete medium (DMEM supplemented with 10% FBS) and incubated overnight at 37°C. On day 2, the culture medium was removed and replenished with a mixture of complete medium (2 ml) with Polybrene (Santa Cruz Biotechnology) at a final concentration of 2 μg/ml. The cells were then infected with 20 μl of the shRNA lentiviral particle stock (Santa Cruz Biotechnology) or control shRNA lentiviral particle stock (Santa Cruz Biotechnology). The infection was performed at 37°C overnight. On day 3, the culture medium was aspirated and replaced with 2 ml of fresh complete medium. Stable clones expressing the PARG shRNA were selected by puromycin at 2.5 μg/ml and validated by Western blotting with anti-PARG antibody (Cell Signaling Technology).