Hbe cells

Study subjects were patients with or without COPD scheduled for bronchoscopies for small nodules or a small amount of bloody sputum at Wuxi People’s Hospital. After using 20 mL of normal saline to lavage the second- and third-generation bronchi, 5 consecutive brushings (Anrei, China) were sampled from the bronchial mucosa. After each brush, cells were harvested into a tube containing 5 mL of RPMI 1640 (Gibco, USA) with 1% penicillin-streptomycin (P/S). Approximately 25 mL of liquid was filtered through a cell strainer (Falcon, USA). After 5 min of centrifugation at 3000 rpm, the cell pellets were washed twice with 2 mL of RPMI 1640, and the final cell pellets were resuspended in BEGM (Lonza, USA). Primary human bronchial epithelial cells were harvested after 7 to 25 days of culture.
HBE cells (ATCC, USA) were maintained in DMEM (Gibco, USA) containing 10% FBS and 1X P/S under 5% CO₂ at 37 °C. When HBE cells reached a confluence of approximately 5×10⁵ per well, the medium was replaced with serum-free high-glucose DMEM for 24 h. Nrf2-overexpressing HBE cells were obtained by transfection with an NFE2L2 overexpression (OE) lentiviral vector. HBE cells were treated with 10 μM fer-1 (Sigma-Aldrich, USA) and 1 μM RSL3 (Sigma-Aldrich) for 1 h before they were exposed to 5% CS extract (CSE) for 72 h.

Human bronchial epithelial (HBE) cells, obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Thermo Fisher Scientific) with 10% fetal bovine serum (FBS; Gibco) at 37°C with 5% CO₂. Normal human lung fibroblasts (NHLFs) were purchased from Cambrex Bio Science Walkersville (Walkersville, MD, USA) and cultured in Dulbecco's Modified Eagle Medium (Gibco) containing 10% FBS, 100 U/mL streptomycin, and 100 μg/mL penicillin (Gibco).