Hexadimethrine bromide h9268

Passage 2 NHBEC were plated in a 6‐well plate at 5 × 10⁴ cells per well and grown overnight. These were purchased heterogeneous NHBEC and not clonal cells. The media was replaced with 800 μL of BEGM with 2 μg/mL of polybrene (Hexadimethrine bromide H9268 Sigma) and 6.25 μL of lentivirus or 12.5 μL of retrovirus stock solutions (minimal concentrations to give >90% transfection determined empirically). The plates were incubated at 37°C for 6.5 h with gentle rocking of the plate every 30 min for the first 2 h. The viral mixture was removed and replaced with BEGM. After 72 h, selection media was added to the wells with 300 ng/mL puromycin for the lentiviral‐infected cells and 600 ng/mL blasticidin for the retroviral cells. Cells with antibiotic resistance after 6 days of selection were considered to have been stably infected with the appropriate virus expressing BMI‐1 or hTERT.

MG132 (M7449), Cycloheximide (C7698), and Hexadimethrine bromide (H9268) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal antibodies against Flag (Sigma-Aldrich, St. Louis, MO, USA), HA (Promotor Biotechnology, Wuhan, China), and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal antibodies against HA (Sigma-Aldrich); rabbit anti-ERK1/2 and p-ERK1/2 (Cell Signaling Technology, Danvers, USA); rabbit anti-USP21 and rabbit anti-MEK2 (Proteintech Biotechnology, Wuhan, China). Anti-Flag affinity agarose, anti-HA affinity agarose (Selleck, Houston, USA).