Trans lentiviral packaging plasmid mix

OECM1 cell line with stable knockdown of BMI1 (OECM1‐shBMI1) was generated by lentiviral expression of a short hairpin RNA (shRNA) specifically targeting BMI1 (shBMI1) as described previously (Yang et al., 2012). OECM1 cells with stable expression of a scrambled nontargeting shRNA (OECM1‐shCtrl) served as a control. Fadu cells with stable overexpression of BMI1 (Fadu‐BMI1) or the control empty vector pCDH‐CMV‐MCS‐EF1‐Puro (Fadu‐CDH) were generated as follows. First, pseudotyped lentiviral particles were produced in 293T cells by cotransfecting pCDH‐BMI1 or pCDH‐CMV‐MCS‐EF1‐Puro with Trans‐Lentiviral Packaging Plasmid Mix (Open Biosystems, Lafayette, CO, USA). Pseudoviral particles were harvested 48 h after transfection and concentrated using PEG‐it Virus Precipitation Solution (System Biosciences), following the manufacturer's instructions. Next, Fadu cells were transduced with the prepared virus in the presence of polybrene (4 μg·mL⁻¹). Two days post‐transduction, cells were split and selected by puromycin (1 μg·mL⁻¹) for 10 days. The stable overexpression of BMI1 was verified by western blotting analysis.

Pseudotyped lentiviruses were produced in 293T cells by co-transfecting lentiviral expression constructs and Trans-Lentiviral Packaging Plasmid Mix following the manufacturer’s instructions (Open Biosystems). 48 hours post transfection, the pseudoviral particles were harvested and concentrated using PEG-it Virus Precipitation Solution (System Biosciences). A549 cells were transduced with prepared lentivirus in the presence of polybrene (4 μg/ml) for two days.