Human recombinant epidermal growth factor and bovine pituitary extract

Human kidney proximal tubular cell line (HK-2) was purchased from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured at 37°C in a normal atmosphere of 95% air and 5% CO₂ in keratinocyte-serum free medium supplemented with human recombinant epidermal growth factor and bovine pituitary extract (Life Technologies, Carlsbad, CA). Hypoxia-reoxygenation was induced in tubular cells to simulate ischemia-reperfusion injury as described in previous studies (30 (link), 31 (link), 36 (link)). In brief, hypoxia was induced in tubular cells by incubation for 2 h in a modified Krebs buffer (137 mM NaCl sodium chloride, 15.8 mM KCl potassium chloride, 0.49 mM MgCl₂ magnesium chloride, 0.9 mM CaCl₂ calcium chloride, 4 mM HEPES) supplemented with 10 mM 2-deoxyglucose, 20 mM sodium lactate, 12 mM KCl potassium chloride and 1 mM sodium dithionite (pH 6.4) in a hypoxia chamber (Billups-Rothenberg, Inc., Del Mar, CA) containing 95% N₂/5% CO₂ at 37°C. After 2 h hypoxia, the modified Krebs buffer was replaced with keratinocyte-serum free medium and cells were cultured for 48 h to 72 h in 95% air and 5% CO₂. Cells without induction of hypoxia were used as a control.

The human OSCC cell lines SCC9, SCC15, and CAL27 were obtained from ATCC (Rockville, MD, USA). UM1, UM2 and SCC1 were provided by Dr. Xiaofeng Zhou (University of Illinois at Chicago, IL, USA). HSC3, HSC6, HN6, CAL33 and normal oral keratinocytes (NOK) were kindly provided by J. Silvio Gutkind (NIH, Bethesda, MD, USA). UM1, UM2, SCC9, SCC15 and SCC25 cell lines were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F12 (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco). SCC1, HSC3, HSC6, HN6, CAL27 and CAL33 cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% FBS (Gibco). NOK cells were grown in keratinocyte serum-free medium containing human recombinant epidermal growth factor and bovine pituitary extract (Life Technologies). All cells were incubated at 37°C in a humidified atmosphere with 5% CO₂. All cell lines were routinely tested for Mycoplasma with PlasmoTest^TM Mycoplasma contamination detection kit (InvivoGen).

VK2 (catalog no. CRL-2616; ATCC) were grown in keratinocyte serum-free medium (KSFM) supplemented with human recombinant epidermal growth factor and bovine pituitary extract (Life Technologies), and bladder epithelial cells (HTB-9; catalog no. 5637; ATCC) were grown in Roswell Park Memorial Institute 1640 (RPMI 1640, Gibco) medium with 10% fetal bovine serum. Cell lines were cultured at 37°C with 5% CO2 and passages 5 to 25 were used for experiments.