L glutamine

The methods used for culturing human DPCs have been described previously [31 (link), 32 (link)]. DPCs (ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 10 ng/mL basic fibroblast growth factor (Peprotech, London, UK) and antibiotic/antimycotic solution (Gibco). Primary human LECs and BECs isolated from foreskin were cultured as described previously [33 (link)]. LECs were cultured on 10 μg/mL fibronectin (Millipore, Billerica, MA, USA)-coated dishes in endothelial cell basal medium (EBM; Lonza, Walkersville, MD, USA), supplemented with 20% FBS, 2 mM l-glutamine (Gibco), antibiotic-antimycotic solution, 10 μg/mL hydrocortisone (Sigma-Aldrich) and 25 μg/mL cAMP (Sigma-Aldrich). BECs were cultured on 10 μg/mL fibronectin-coated dishes in EBM supplemented with 20% FBS, 2 mM l-glutamine, 1x antibiotic-antimycotic solution and 0.4% endothelial cell growth supplement (ECGS; PromoCell). Human dermal fibroblasts (DFs) were cultured in DMEM supplemented with 10% FBS and antibiotic/ antimycotic solution. Cells were incubated at 37°C in a 5% CO₂ incubator.

Non-tumor, non-cystic fibrosis human bronchial epithelial cell line immortalized with Human Papilloma Virus-18 E6 and E7 oncogenes^{75 (link)} was generously provided by Dr. A. K. Bauer (University of Colorado, USA). HBE1 cells retain ion transport and other properties comparable to the native cells^{75 (link)}. HBE1 cells were cultured in DMEM/F12 (Cat. No. L0094, Biowest, Nuaillé, France) medium supplemented with 4 µg/mL human insulin, transferrin (5 µg/mL), human epidermal growth factor (10 ng/mL), dexamethasone (0.1 µM), cholera toxin (20 ng/mL), Plasmocin^TM (2.5 µg/mL, Invivogen, San Diego, CA), L-glutamine (2.5 mM), endothelial cell growth supplement (0.1% v/v, Promocell, Heidelberg, Germany) (pH: 7.40). For the experiments, HBE1 cells were plated in a density of 50,000 cells/cm². into a 24 well-plate (0.5 mL/well). 48 hrs after cell seeding, the growth medium was exchanged with the exposure medium (culture medium with a diluted toxicant; TPA) and the cells were exposed for 24 h.