Keratinocyte serum free media

The esophageal epithelial cell line (human telomerase reverse transcriptase-immortalized EPC2 cell line) was a kind gift from Dr. Anil Rustgi (University of Pennsylvania, Philadelphia, PA).^{42 (link)-44 (link)} EPC2s were cultured in Keratinocyte Serum-Free media (Life Technologies) supplemented with bovine pituitary extract, epidermal growth factor, and amphotericin (Life Technologies). For differentiated EPC2 cell cultures, EPC2s were grown to confluence for 2 days while being fully submerged in low-calcium ([Ca²⁺] = 0.09 mM) supplemented Keratinocyte Serum-Free media on 0.4 μm pore-size permeable supports (Corning Incorporated, Corning, NY). Confluent monolayers were then switched to high-calcium ([Ca²⁺] = 1.8 mM) media for an additional 5 days. The culture medium was removed from the inner chamber of the permeable support in order to expose the cell monolayer to the air interface at day 7. Differentiated EPC2s were analyzed 7 days after ALI induction.

The cell lines RWPE-1, LNCaP, 22Rv1, PC3, and VCaP were obtained from American Type Culture Collection (Manassas, VA). LNCaP, 22Rv1 and PC3 were routinely cultured in RPMI-1640 (Corning Inc., Corning, NY), supplemented with 1% penicillin/streptomycin, 1% l-glutamine (Corning Inc.), and 10% fetal bovine serum (FBS, R&D Systems, Minneapolis, MN) unless otherwise noted. RWPE-1 were cultured in Keratinocyte serum-free media (Corning Inc.) containing recombinant epidermal growth factor and bovine pituitary extract as well as 1% penicillin/streptomycin. VCaP were cultured in DMEM + GlutaMAX (Corning Inc.) containing 10% FBS and 1% anti-mycotic antibiotic (Corning Inc.). The C4-2B cell line was a gift from Dr. Conor Lynch (Moffitt Cancer Center, Tampa, FL), and was cultured in DMEM (Corning Inc.), under the same conditions as LNCaP and 22Rv1. All cell lines were authenticated by STR profiling (Labcorp, Burlington, NC) and routinely tested for mycoplasma (LookOut Mycoplasma PCR Detection Kit, Sigma Aldrich, St. Louis, MO). Cell lines were passaged fewer than 10 times. Trypsin was purchased from Corning Inc. IBMX, Tolvaptan, forskolin, desmopressin (dAVP) and H89 were purchased from Tocris (Bristol, UK). Phenytoin was purchased from Sigma Aldrich. Relcovaptan was purchased from Axon Medchem (Reston, VA).