Esophageal epithelial cell culture and TGF-β1 treatment

EPC1-hTERT and EPC2-hTERT, established from independent primary cultures of normal human esophageal epithelial cells, and their derivatives were grown in Keratinocyte-serum free medium (Invitrogen, Carlsbad, CA) at 37°C in a 5% CO₂ atmosphere as described previously (18 (link), 20 (link), 22 (link)–23 (link)). HCE7, an ESCC cell line was grown as described previously (24 (link)). Countess^™ Automated Cell Counter (Invitrogen) was used to count cells with 0.2% Trypan Blue dye to exclude dead cells. Cells were treated with 5 ng/ml of recombinant human TGF-β1 (R&D Systems, Minneapolis, MN) reconstituted in 4 mM HCl containing 0.1% bovine serum albumin. AG 1478 (Calbiochem, La Jolla CA) was reconstituted in dimethyl sulfoxide and used at 100 nM. Phase contrast images were acquired using a Nikon Eclipse TS100 microscope. Spindle-shaped cells were scored by counting at least 100 cells per high-power field (n=6) under light microscopy.