Gw4064

GCDCA (Purity 97%) was purchased from Shanghai Yuanye Bio-Technology Company (Shanghai, China). It was dissolved in cell medium and stored at 4 °C before used. LDN-193189 and GW4064 were purchased from Selleck Chemicals Company (Houston, TX, USA). Guggulsterone was purchased from MedChemExpress Company (Shanghai, China). LDN-193189, GW4064 and guggulsterone were dissolved in DMSO and stored at −20 °C before used.

We isolate hepatocytes from yellow catfish liver tissues according to our recent publication [21 (link)]. The isolated cells were seeded onto 25 cm² plates (1 × 10⁶ cells/mL) and placed in an incubator at 28 °C and 0.5% CO₂ (Sanyo, Japan). Two pathway inhibitors and two agonist were used in the in vitro trials. They are rapamycin (RM, autophagy agonist, Selleck, USA), 3-methyladenine (MA, autophagy inhibitor, Selleck, USA), GW4064 (GW, FXR agonist, Selleck, USA) and guggulsterone (GS, FXR inhibitor, Sigma, USA), respectively. Treatment groups were designed as follows: control, 0.5 mM FA (PA, palmitic acid: OA, oleic acid =1:1), 20 nM rapamyicn (RM), 5 mM MA, 20 μM guggulsterone (GS), 1 μM GW4064 (GW), 0.5 mM FA + 20 nM rapamycin (FA + RM), 0.5 mM FA + 5 mM MA, 0.5 mM FA + 1 μM GW4064 (FA + GW) and 0.5 mM FA + 20 μM guggulsterone (FA + GS), respectively. Each treatment had three replicates. Based on our pilot trials, the FA level was set at a concentration that did not adversely affect cell viability. We selected the inhibitors/agonist concentrations based on our other pilot trials and to previous reports [22 (link), 23 (link)]. The cells were collected at 48 h for the analyses described below.