2.3. Cell lines, cell culture, transfection, virus infection and treatments

The 786‐O, OSRC‐2, SN12‐PM6, ACHN, and HEK293T cell lines were purchased from the Cell Bank of Shanghai Institute of Cell Biology (Chinese Academy of Medical Science, Shanghai, China). These cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, HyClone, Logan, Utah, USA) with 10% fetal bovine serum (HyClone) in the presence of 5% CO₂ at 37°C in a humidified incubator (Thermo Fisher Scientific). Transfection was performed using Lipofectamine™2000 (Thermo Fisher Scientific). For lentiviral infection, HEK293T cells were transfected with packaging vectors (pRSV‐Rev, pMD2.G, and pCMV‐VSV‐G) and LacZ shRNA, JUP shRNA, HIF2α shRNA plasmids, or psi‐HA‐JUP plasmids; the viruses were collected 60 h after transfection and used to infect 786‐O or ACHN cells. Stably transduced cells were screened using puromycin (Sigma) at a final concentration of 2 mg/mL for 3 days. Cells were treated with 10 μg/mL MG132 (S1748, Beyotime Biotechnology, Beijing, China) for 6 h before being harvested for immunoblotting with anti‐HIF2α antibody or control antibodies.