Modulation of Cellular Pathways by Bile Acids

LCA (cat # L6250; Sigma-Aldrich, St. Louis, MI, USA) was dissolved in DMSO at a stock concentration of 100 mM. LCA was used at a concentration of 0.03 µM, corresponding to normal human serum concentration [7 (link), 47 , 48 (link)]. Non-treated cells received 0.001% DMSO in the medium as a vehicle. Glutathione (GSH; cat # G4251; Sigma-Aldrich) was used at a final concentration of 5 mM. Pegylated catalase (pegCAT; cat # C4963; Sigma-Aldrich) was used at a concentration of 500 U/ml. BA receptor antagonists (NF449 [95 (link)], CINPA1 [96 (link)], DY268 [97 (link)], and GSK2033 [98 (link)]) were acquired from Tocris Bioscience (Bristol, UK), and ketoconazole [99 (link)] was purchased from Sigma-Aldrich. NF449 (G_sα-selective antagonist; 5 µM) was used to inhibit TGR5 signaling. Nuclear receptor activation was inhibited using 5 µM CINPA1 (CAR antagonist), DY268 (FXR antagonist), and GSK2033 (LXR antagonist). PXR downstream signaling was inhibited using ketoconazole (5 μM). Chemotherapy drugs (irinotecan, paclitaxel, gemcitabine, 5-fluorouracil and oxaliplatin) were purchased from Sigma-Aldrich. Silencer Select siRNAs targeting TGR5 (GPBAR1-siRNA ID: s195791), VDR (NR1I1- siRNA ID: s14777), and FXR (NR1H4-siRNA ID: s19371), and the negative control siRNA #1 (cat # 4390843) were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and used at a final concentration of 30 nM.