Pcdna 3.1 vector

All cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). ACHN, A498, and HK-2 cell lines were cultured in MEM medium, while 786-O and 769-P cell lines were cultured in RPMI1640 medium (Gibco, NY, USA). The medium was supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA), 100 μg/ml streptomycin, and 100 U/ml sodium penicillin (Biotechnology, Beijing, China). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂. The control overexpression plasmid (Vector) and the DBT overexpression plasmid (OE-DBT) were constructed using the pcDNA 3.1(+) vector (Hanheng Biotechnology, Shanghai, China). A498 cells were transfected with the OE-DBT and Vector plasmids using Lipofectamine 3000 transfection reagent (Invitrogen, Waltham, Massachusetts, USA), following the manufacturer’s instructions.

For stable transfection of short hairpin RNA (shRNA), predesigned shRNA-expressing lentivirus particles were synthesized (GeneChem, Shanghai, China) using the following shRNAs: shPHB2 #1 forward: 5′- CCAGAATATCTCCAAGACGAT-3′ and reverse: 5′-ATCGTCTTGGAGATATTCTGG-3′; shPHB2 #2 forward: 5′-GCTGAGCTTTAGCCGAGAGTA-3′ and reverse: 5′-TACTCTCGGCTAAAGCTCAGC-3′; and shControl (shCtrl), forward: 5′-TTCTCCGAACGTGTCACGT-3′ and reverse: 5′-ACGTGACACGTTCGGAGAA-3′. For overexpression of PHB2, a full-length cDNA encoding PHB2 whose C-terminus was fused with a cDNA fragment encoding flag was inserted into the pcDNA3.1 vector (Hanheng Biotechnology, Shanghai, China). Lentiviral infection of A549 and H1299 cells was performed according to the manufacturer's protocols. Briefly, cells were infected with the lentivirus for 48 h and selected with puromycin by increasing the concentration from 1 to 3 ng/mL for 1 week. The surviving cells were cultured for further research.