NOX4 short-hairpin RNAs (shRNAs) or scrambled control shRNA was designed, synthesized and packaged into lentivirus particles (Corues Biotechnology, Nanjing, China). Cells were plated into six-well plates (3 × 105 cells per well). Before transfection, the culture medium was replaced with DMEM with 10%FBS and 1 µg/mL Polybrene (GeneChem, Shanghai, China). Infectious lentivirus particles were harvested for 72 h after transfection.
To establish NOX4-overexpressed HPAC cells, HPAC cells were transfected with NOX4 Human Tagged ORF Clone (RC208007) and the pcmv6 empty vector (OriGene, MD, USA) using lipo3000 (Thermo Fisher Scientific, MA, USA). After 72 h, cells were treated with G418 (Sigma-Aldrich Corp., MO, USA) to select stably transfected clones.
To establish NOX4-overexpressed HPAC cells, HPAC cells were transfected with NOX4 Human Tagged ORF Clone (RC208007) and the pcmv6 empty vector (OriGene, MD, USA) using lipo3000 (Thermo Fisher Scientific, MA, USA). After 72 h, cells were treated with G418 (Sigma-Aldrich Corp., MO, USA) to select stably transfected clones.