Chenodeoxycholic acid cdca

Obeticholic acid (OCA) was provided by Intercept Pharmaceuticals, Inc. San Diego, USA, and was prepared as stock solution in dimethyl sulfoxide (DMSO, CAS Number 67-68-5, Sigma-Aldrich, Milan, Italy) and then diluted (1:10⁵) in culture medium at the desired final concentration; the same amount of DMSO was added to controls. Stock solutions of OCA were freshly prepared every 15 days.
Gemcitabine hydrochloride (Sigma-Aldrich, CAS Number: 95058-81-4) was prepared as a stock solution in water and added in the culture media of iCCA cells after dilution (1:10⁴). Cisplatin (Sigma-Aldrich, CAS Number: 15663-27-1) was prepared as a stock solution in DMSO and added to cell cultures, after appropriate dilution (1: 10⁵) in H69 medium. We used the concentration of Gemcitabine (10 μM) and Cisplatin (20 μM) previously demonstrated to exert maximal inhibitory effects on cell proliferation in the same primary human iCCA cell cultures used in the present study [19 (link)].
Chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) were purchased from Sigma-Aldrich, diluted in DMSO and added into culture medium after dilution to reach the desired final concentration. The same amount of DMSO was added to controls.
During the experiments, the cell medium was refreshed every 3 days.

Sodium alginate (Low viscosity SA, 99%), chenodeoxycholic acid (CDCA, 99%) and poly-l-ornithine hydrochloride (PLO; molecular weight 30–70 kDa) were purchased from Sigma chemical Co, (St. Louis, MO, USA). Calcium chloride dehydrate (CaCl₂·2H₂O, 98%) was obtained from Scharlab S.L, Australia. Dulbecco’s modified Eagle’s medium (DMEM) and other required supplements were purchased from Sigma Chemical Co (St. Louis, MO, USA). All other solvents and reagents were purchased from Merck (Bayswater, VIC, Australia) and were HPLC garde and used without further purification.

HepG2/C3a were provided by the ATCC. HepG2/C3a cells were grown in MEM media supplemented with 10% FBS and 1% antibiotics-antimycotic at 37 °C in a humidified incubator and with an atmosphere of 5% CO₂. Cells were exposed to Cu at the indicated concentrations and for the indicated times. When required, chenodeoxycholic acid (CDCA, Sigma-Aldrich, Saint Quentin Fallavier, France) at 80 µM or the equivalent volume of DMSO was added for the last 17 h of exposure.

Dulbecco's Modified Eagle Medium (DMEM), phosphate‐buffered saline (PBS), fetal bovine serum (FBS), 0.25% (w/v) trypsin–EDTA and penicillin–streptomycin (P/S) were obtained from Gibco (Gaithersburg, MD, USA). Calf serum (CS) was supplied by HyClone (Logan, UT, USA). GW4064 and chenodeoxycholic acid (CDCA) was purchased from Sigma (St Louis, MO, USA). Pierce™ BCA Protein Assay Kit, Lipofectamine 3000 Reagent and SuperSignal West Femto Maximum Sensitivity Substrate were purchased from Thermo Fisher Scientific (Grand Island, NY, USA). The mouse antibody against FXR was purchased from R&D, and the antibodies against pro‐caspase‐3 and cleaved caspase‐3 were obtained from Cell Signalling (Danvers, MA, USA). The antibodies against TonEBP, COX‐2, SMIT, and Caspase‐3 Assay Kit (Fluorometric) were offered by Abcam (Cambridge, MA, USA). Expression vectors of pcDNA, hFXR and NF‐κB (p65) were obtained from Origene (Beijing, China). Triton X‐100 was offered by Bio‐Rad (Hercules, CA, USA).