For hypoxia, NES cells were incubated at 1% oxygen by nitrogen in an INVivo2 400 (LAF Technologies, Queensland, Australia) hypoxia work station. For individual experiments, cells were cultured in neutral medium (pH7) alone or with periodic exposures to acidic medium (pH 5.5) containing a mixture of conjugated bile acids (glycocholic acid, glycodeoxycholic acid [both from Calbiochem, San Diego, CA], taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, and taurodeoxycholic acid [Sigma-Aldrich, St. Louis, MO] in a 20:6:3:15:3:1 molar concentration, total concentration 400 μM) as previously described. 14 (link), 16 (link)-18 (link) Neutral or acidic bile salt medium was added for 10, 30 or 60 minutes, then replaced with growth medium. These exposure durations were chosen to simulate typical episodes of gastrooesophageal reflux. 19 (link), 20 (link) All assays were performed over a 24-hour time course unless otherwise indicated.
Additional details are provided in the Supplemental Material and Methods section and in Supplemental Tables 1 and 2.