OE-33 and OE-21 cell lines were derived from ADE and ESCC, respectively, and were obtained from ATCC. Both lineages were cultured in RPMI medium (Invitrogen) supplemented with 10% fetal bovine serum (Gibco) and 1% of the cocktail penicillin/glutamine/streptomycin (Invitrogen) and maintained at 37 °C, under 5% CO2. The immortalized normal esophageal epithelium cell line, Het1a, was also obtained from ATCC, and was cultured in BEBM plus supplements (Lonza) and maintained at 37 °C under 5% CO2. The three cell lines used in this study were authenticated by using STR (or SNP) profiling within the last 3 years and regularly tested for mycoplasma contamination, by using MycoSensor PCR Assay Kit (Agilent). Cells were stimulated with Oleic Acid (OA—1, 2 and 5 μM) for 24 h or untreated. Het1a cell line was incubated with deoxycholic bile acid (DCA—200 μM) or acidified medium (acid pH 5.0), or the combination of both, in plain medium at 37 °C under 5% CO2, for variable number of 3 min pulses, with 30 min interval between them. Additionally, Het1a cells were treated with increasing amounts of nicotine-derived nitrosamine ketone (NNK—0.5, 1.0 and 2.0 μM) and increasing concentrations of ethanol (EtOH 0.01, 0.1 and 1.0%) for 24 h or untreated.

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