Plpcx plasmid

HCT116 cells were co-transfected with Control CRISPR/Cas9 (sc-418922), p27 CRISPR/Cas9 KO (sc-400074) or PCAF CRISPR/Cas9 KO (sc-400753) plasmids from Santa Cruz and with pLPCX plasmid (K1061-1, Clontech) using Lipofectamine 2000 transfection reagent (Invitrogen P/N 52887) according to manufacturer's protocol. Transfected cells were subsequently selected with media containing 2 mg/ml of Puromycin (Sigma Aldrich) for 3–5 days. Successful transfection was confirmed by WB with specific antibodies. For the stable generation of a p27KO cell line we used the CRISPR/Cas9 system transfecting HCT116 cells with p27 Double Nickase Plasmid (sc-400074-NIC) and with Control Double Nickase (sc-437281) for the control cell line. Individual GFP positive colonies were isolated by cell sorting (FACSAriaII) and seeded in 96-well plates and grown for 2–3 weeks until the positive colonies reached confluence when they were transferred to 24-well plates. When the latter reached confluence, cells were trypsinized and an aliquot was kept for protein screening by WB and mRNA levels validation. The remaining cells were frozen or expanded in 60 mm dishes for additional analyses.