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.