Bronchial epithelial cell medium

The immortalized cell line, 16HBE14o-, was derived from bronchial surface epithelial cells [14] (link). Standard culture techniques were used as described previously [1] (link), [13] (link). In brief, cells were maintained in Minimum Essential Medium with Earle’s salts supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were cultured on plastic flasks coated with fibronectin and collagen (BD Biosciences, San Jose, CA, USA) and were incubated in humidified 95% air-5% CO₂ at 37°C. Primary human bronchial epithelial (HBE) cells were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and maintained in Bronchial Epithelial Cell Medium (ScienCell Research Laboratories). All other general laboratory reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), and all cell culture reagents were obtained from Invitrogen (Carlsbad, CA, USA). Before the use of UDP, this nucleotide (10 mM) was incubated (1 h at 37°C) in HEPES-buffered saline containing hexokinase (10 IU/ml; Boehringer, Mannheim, Germany) and 22 mM D-glucose in order to remove contaminating nucleotide triphosphates. The resulting solution was then aliquoted and stored at −20°C [15] (link).

Human bronchial epithelial cells BEAS-2B were acquired from American Type Culture Collection (Manassas, VA, USA) and grown in a bronchial epithelial cell medium (ScienCell, CA, USA) supplement with 10% Fetal bovine serum (FBS) (Hyclone, UT, USA) and 1% antibiotics (Invitrogen, CA, USA) at 37°C [14 (link),15 (link)]. Cells in the logarithmic growth phase were trypsinized and seeded into 6-well plates, 35-mm culture dishes or 96-well plates according to the appropriate assay conditions. The cell injury model was established by stimulating with LPS (Sigma-Aldrich, MO, USA) at various concentrations (0, 0.25, 0.5, 1, and 2 μg/ml) for 24 h [14–17 (link)].

Human bronchial epithelial cells (16HBE cells) were from the Chines Academy of Sciences (Shanghai, China). 16HBE cells were cultured in bronchial epithelial cell medium (ScienCell Research Laboratories, Inc.) and incubated at 37°C in a humidified atmosphere containing 5% CO₂. And then, 16HBE cells were seeded into a 6‐well plate and serum deprivation for 12 hours in bronchial epithelial cell medium, PM2.5 was suspended and sonicated in sterile saline to a final concentration of 1 mg/mL, and 16HBE cells were treated with 20 μg/mL PM2.5 for 24 hours.

HBEpCs were cultured in Bronchial Epithelial Cell Medium (ScienCell) or RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in an incubator with 5% CO₂. Cells at passages 4–6 were used for subsequent experiments. For LPS treatment, cells were cultured in media containing 0, 5, and 10 ng/ml LPS for 48 h. For 5-azaCytidine (5-azaC, Sigma-Aldrich) treatment, HBEpCs were incubated in media containing 1, 10, or 100 nM 5-azaC for 24 h.

Human bronchial epithelial (HBE) cells were from Sciencell Company and grown in bronchial epithelial cell medium (ScienCell, 3211). Non-small cell lung cancer (NSCLC) cell lines (H1299, H292, A549) were from ATCC and SPC-A1 cell line was a gift from Dr. Xuerong Wang’s group (Department of Pharmacology, Nanjing Medical University). NSCLC cell lines were grown in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco). All cells were cultured under an atmosphere of 5% CO₂ at 37°C.