Gastric Cancer Cell Line Bile Acid Exposure

For our purposes, we required well-established, acid-stable gastric cancer cell lines with comparable levels of c-Myc expression (10 (link)). Accordingly, we purchased AGS (ATCC^® CRL-1739^™) and NCI-N87 (ATCC^® CRL-5822^™) cell lines from the American Type Culture Collection (Manassas, VA, USA). These gastric cancer cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) (GIBCO Invitrogen) containing 4.5 mg/l glucose, 100 mg/l streptomycin, and 2 mM L-glutamine supplemented with 10% fetal bovine serum (FBS) (GIBCO Invitrogen). They were maintained at 37°C under a humidified 5% CO₂ atmosphere in a CO₂ incubator (Sanyo). Solutions of bile acids (Sigma-Aldrich) were prepared using appropriate solvents according to the manufacturer's protocols (Table SI). AGS and NCI-N87 cells were cultured in the growth medium for 24 h and then transferred to fresh, serum-free medium containing 100 µM of a bile acid for 48 h, with the bile acid being cholic acid (CA; Sigma-Aldrich, C9377), chenodeoxycholic acid (CDCA; Sigma-Aldrichl, C1129), taurocholic acid (TCA; Sigma-Aldrich, T4009), or glycochenodeoxycholic acid (GCDCA; Sigma-Aldrich, G0759). Afterward, we extracted the total RNA and total protein from the cells.