In vitro model of alveolar epithelial cells

The A549 cell line, which is derived from a human alveolar cell carcinoma, has many properties of human alveolar epithelial cells and was utilized because it is the most widely used in vitro model of type II pulmonary alveolar epithelial cells [38 (link)]. The cells were obtained from the American Type Culture Collection (ATCC^® CCL-185^TM) and were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% glutamine in a humidified atmosphere with 5% CO₂ at 37 °C. Before the experiments, the cells were authenticated by short tandem repeat profiling and tested for mycoplasma contamination. For H/R conditions, Cells were exposed to a microaerophilic system (Thermo Fisher Scientific 8000, Marietta, GA) with 5% CO₂ and 1% O₂ balanced with 94% N₂ at 37 °C for 6 h followed by reoxygenation in 95% air and 5% CO₂ for 2 h to establish H/R conditions, as described in previous reports with a few modifications [28 (link)]. For 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP, Selleck), nicotinamide (NAM, Sigma-Aldrich) or trichostatin A (TSA, Sigma-Aldrich) treatment, the cells were incubated with 50 μM 3-TYP, 10 mM NAM or 0.5 μM TSA for the indicated times and then subjected to H/R, as needed [23 (link)].