Generating Stable Knockdown Cell Lines

Mission short hairpin RNA (shRNA) lentiviral vectors expressing five ATR or p62-specific shRNAs (constructs 1 to 5) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). The vectors were packaged into capsids in 293T cells, and lentiviral particles were prepared as previously described (44 (link)). CIN612 HPV-positive cells were then transduced with these lentiviral soups overnight at 37°C. The viral soups consist of concentrated shRNA or scrambled shRNA lentiviruses and 4 μg/ml hexadimethrine bromide (Polybrene; Sigma-Aldrich, St. Louis, MO) in 5 ml E medium. The incubated cells were changed to fresh E medium the next day for an additional 48 h before further processing or analysis. Stable knockdown lines were selected by coexpressed puromycin drug resistance markers.