Epidermal growth factor (egf)

For the alveolar cell differentiation, cells with 10⁵ cells had seeded on the collagen I-coated 24 well plate. MCDB201 medium (Sigma-Aldrich) supplemented with 5% FBS, Insulin-Transferrin-Selenium (Gibco), 1X penicillin-streptomycin-amphotericin B solution (PSA from 100X stock, Biological Industries), and 25 ng/mL of epidermal growth factor (Corning) were replaced every two days. Cells had differentiated after 7-14 days of culture. For trachea epithelial cell differentiation, cells with 10⁵ cells had seeded on the collagen I-coated 24-well transwell inserts with 0.4 μm pore (Corning). MCDB201 medium supplemented with 5% FBS, Insulin-Transferrin-Selenium, 1X PSA, 0.1 μg/mL of cholera toxin, 30 μg/mL of bovine pituitary extract (Gibco), and 25 ng/mL of epidermal growth factor. Medium in the transwell inserts with 0.4 μm pore and the lower chambers had replaced every two days. As cells reached 100% confluence, cells had maintained under the air-liquid interface by leaving the transwell inserts empty for 10-15 days.

DMEM/F12, penicillin-streptomycin, fetal bovine serum, TRIzol reagent, SYBR Green, and RIPA buffer were supplied from Thermo Fisher Scientific (Waltham, MA, USA). Epidermal growth factor, and ITS (the mixture of insulin, transferrin, and selenious acids) were obtained from Corning Incorporated (New York, NY, USA), dimethyl sulfoxide, and genistein (synthetic product, purity ≥ 98%) were provided by Sigma-Aldrich (St. Louis, MO, USA). ROS assay kit was obtained from Beyotime Biotechnology (Shanghai, China). The cell counting kit (CCK-8) was bought from Med Chem Expression (Princeton, NJ, USA). The malondialdehyde (MDA), glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) assay kits were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The Trans Script First-Strand cDNA Synthesis Kit was supplied by Trans Gen Biotech (Beijing, China). The VDF membranes, and ECL agent were provided by Bio-Rad Laboratories, Incorporated (Irvine, CA, USA).

Intestinal porcine epithelial cells (IPEC-J2; ACC 701, Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Culture, Braunschweig, Germany) were cultivated and routinely maintained using DMEM/F12 (1:1) without L-glutamine (Pan Biotech, Aidenbach, Germany), adjusted to 2.4 g/L NaHCO₃ (Pan Biotech) and supplemented with 1% insulin–transferrin–selenium ITS (Gibco/Life Technologies, Thermo Fisher Scientific, Vienna Austria), 2.5 mM Glutamax (Gibco/Life Technologies, Thermo Fisher Scientific), 5 ng/mL epidermal growth factor (Corning Inc, Corning, USA), and 16 mM HEPES (Merck/Sigma Aldrich, Vienna, Austria). The medium was further supplemented with 10% heat-inactivated (30 min at 56 °C) fetal bovine serum FBS (Gibco/Life Technologies, Thermo Fisher Scientific) and 1% penicillin–streptomycin (Merck/Sigma Aldrich) directly before use. Cultures were incubated at 39 °C and 10% CO₂ under a humidified atmosphere (CO₂ incubator, Binder, Tuttlingen, Germany) and sub-cultivated upon exceeding 90% confluency in the cultivation vessels. Routine testing for mycoplasma contaminations was conducted by PCR (Venor^® GEM Classic Mycoplasma Detection Kit, Minerva Biolabs, Berlin, Germany).

IPEC-J2 cells were obtained from Dr. Guoyao Wu's laboratory at Texas A&M University and cultured in Dulbecco's modified Eagle medium/F12 (DMEM/F12, Thermo Fisher Scientific, MA, USA) supplemented with 5% fetal bovine serum (FBS, Thermo Fisher Scientific, MA, USA), 0.1% ITS (5 μg/L insulin, 5 μg/L transferrin and 5 ng/L selenious acid, Corning Incorporated, NY, USA), 0.01% epidermal growth factor (5 μg/L, Corning Incorporated, NY, USA) and 1% pen-strep (Thermo Fisher Scientific, MA, USA) at 37 °C in a humidified atmosphere with 5% CO₂.

The dermal endothelial cell line human microvascular endothelial cells (HMEC-1), was originally a gift from Dr. E. Ades and Dr. T. J. Lawley (Emory School of Medicine and the Centers for Disease Control and Prevention). This cell line is currently available from ATCC. HMEC-1 were cultured in MCDB 131 (Gibco) supplemented with 10 mM L-glutamine, 10 ng/ml epidermal growth factor (Corning), 1 μg/ml hydrocortisone (Sigma), 15% Hyclone Fetal bovine serum (FBS, Thermo), and 25 mM HEPES (Gibco). Two types of primary cells, human dermal lymphatic endothelial cells (HDLEC) and human dermal microvascular endothelial cells (HDMEC), were purchased from ScienCell. These primary cells were cultured in endothelial cell medium (ECM) with the endothelial cell growth supplement (ECGS) and 5% FBS (all from ScienCell), according to the vendor’s protocol. All human cells were grown at 36.5°C under 5% CO₂. For infection, passage of endothelial cells was limited to 20 or less for HMEC-1 and 13 or less for HDLEC and HDMEC. In preliminary experiments, we confirmed the morphological similarity of HDMEC to the cell line HMEC-1. For screening, HMEC-1 was selected for use as a stable microvascular endothelial cell type, and for some experiments was compared to HDLEC, which possesses morphologically well-organized intercellular junctions.