Gw4064

After a midline laparotomy, mice were injected with heparin (100 μg/kg), and an atraumatic clip was used to interrupt arterial/portal venous blood supply to the cephalad liver lobes. After 60 to 90 minutes of ischemia, the clip was removed to initiate liver reperfusion. Sham controls underwent the same procedure but without vascular occlusion. Mice were killed after 6 hours or 5 days of reperfusion, and liver and serum samples were collected. To test the effect on acute liver IRI, GW4064 (5 mg/kg, intraperitoneally; Tocris Bioscience) or OCA (30 mg/kg, oral gavage; Sigma‐Aldrich, St Louis, MO) was administered 1 to 2 hours before the onset of liver ischemia. To test the effect on liver recovery from IRI, GW4064 (5 mg/kg) was administered intraperitoneally at 24 hours, day 2, day 3, and day 4 after reperfusion. Levels of serum alanine aminotransferase (sALT) were measured with an autoanalyzer by IDEXX Laboratories (Westbrook, Maine). A portion of the liver specimens was fixed in 10% buffered formalin and embedded in paraffin. Liver sections (4 μm) were stained with hematoxylin and eosin (HE). The severity of liver IRI was graded blindly using Suzuki's criteria20 on a scale from 0 to 4. Liver sections were processed for immunohistochemical staining of myeloperoxidase (Novus Biologicals) to quantitate polymorphonuclear leukocyte infiltration as described.21, 22

The normal GES-1 human gastric epithelial cell line, AGS, BGC-823 and MKN-45 human gastric adenocarcinoma cell lines, and SGC-7901 human gastric carcinoma cell line were obtained from the Academy of Military Medical Science (Beijing, China) and cultured in RPMI-1640 medium. All cells were maintained at 37°C in a humidified incubator containing 5% CO₂. Chenodeoxycholic acid (CDCA; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany; cat. no. c9377), GW4064 (FXR agonist; Tocris Bioscience, Bristol, UK; cat. no, 2473), and the FXR antagonist guggulsterone (Gug; Sigma-Aldrich; Merck Millipore; cat. no. 1302214) were dissolved in dimethyl sulfoxide. The cells (1–5×10⁵) were seeded in six-well and 6 cm cell culture dish respectively. When the cells reached 80–90% confluence, they were serum-deprived overnight and then treated with CDCA (0, 50, 100 and 200 µM) in the presence or absence of Gug (5, 50 and 100 µM) or GW4064 (0.25, 0.5, 1, 5 and 10 µM) for 24 h under 37°C.

Anti-FXR (sc-13063), anti-ACSL4 (sc-365230), anti-FTH1 (sc-376594), and anti-GAPDH (sc-32233) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), while anti-HMOX1 (43966), anti-caspase8 (4790), anti-caspase3 (9662), anti-cleaved caspase3 (9661), anti-actin (4970), and anti-3-nitro tyrosine (3-NT) (9691) antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-MAFG (PA5-30086) and anti-4-hydroxynonenal (4HNE) (bs-6313R) antibodies were purchased from Invitrogen (Carlsbad, CA, USA) and Bioss (Woburn, MA, USA), respectively. Anti-AIFM2 (LS-C382008-50) antibody was purchased from LSBio (Seattle, WA, USA). GW4064 was obtained from Tocris Bioscience (Bristol, UK). Anti-GPX4 antibody (ab219592) was obtained from Abcam (Cambridge, UK). Erastin was purchased from MedChemExpress. Ferrostatin-1 and cisplatin were purchased from Sigma, Inc (St. Louis, MO, USA). The Image-iT lipid peroxidation kit (C11-BODIPY 581/591) was purchased from Thermo Fisher (Waltham, MA, USA). ON-TARGETplus human short-interfering RNAs against FXR (siFXR, L-003414) and AIFM2(siAIFM2, L-004443) were purchased from Dharmacon, Inc (Lafayette, CO, USA).