Compound c

The Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco (Carlsbad, CA, USA). Galectin-3 and compound C (AMPK inhibitor) were purchased from Abcam (Cambridge, UK). 3-MA was obtained from Sigma Aldrich (St. Louis, MO, USA). Verapamil was purchased from Hefeng Pharmaceutical (Shanghai, China). The primary antibody against phospho-AMPK, AMPK, phospho-mTOR, mTOR and GAPDH were acquired from the Cell Signaling Technology Inc (Danvers, MA, USA). The primary antibodies against TXNIP, LC3B and Runx2 purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Galectin-3, anti-P62, anti-NLRP3, anti-caspase-1, anti-IL-1β, anti-OPN and anti-BMP2 were obtained from Abcam. All other chemicals were from commercial sources.

Telmisartan and valsartan were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). Irbesartan and losartan were purchased from Wako Pure Chemical Industries (Osaka, Japan). Telmisartan was prepared as a 10 mM stock solution in dimethyl sulfoxide (DMSO). Irbesartan and valsartan were prepared as 100 mM stock solutions in DMSO, and losartan was prepared as a 100 mM stock solution in H₂O. The stock solutions were stored at -20°C. Compound C was purchased from Abcam (Cambridge, UK). MK-2206 was purchased from ChemScene Chemicals (Monmouth Junction, NJ, USA). SCH772984 was purchased from Selleck Chemicals (Houston, TX, USA).

Δ⁹-Tetrahydrocannabinol (Δ⁹-THC, kindly provided by Dr. Satoshi Morimoto, Kyushu University) was dissolved in DMSO (FUJIFILM Wako Pure Chemical, Osaka, Japan) at a final concentration of 100 mM and stored at −80 °C until use. Ethanol, AM251, and AM630 were purchased from FUJIFILM Wako Pure Chemical. Tauroursodeoxycholic acid (TUDCA) was from Millipore Corp (USA). 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) was from Sigma-Aldrich (St. Louis, MO, USA). Compound C was from abcam (Cambridge, MA, USA).

Aspc-1, HepG2, Panc02, and H22 cell lines were obtained from the KeyGEN Biotechnology Company (China). HT1080 and SW480 were obtained from the FuHeng BioLogy Company (China). HT1080 cancer cells were cultured in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum (FBS), glutamine (2 mM), penicillin (100 U/ml), and streptomycin (0.1 mg/ml). SW480, Aspc-1, HepG2, Panc02, and H22 were cultured in high Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, L-glutamine (4 mM), and penicillin (100 U/ml) and streptomycin (0.1 mg/ml). All cell lines were maintained in a humidified atmosphere containing 5% CO₂ at 37 °C and tested for mycoplasma prior to the commencement of experiments. Unless otherwise indicated, cell culture medium was changed every 3 days, and cells were passaged using 0.05% trypsin/EDTA. Erastin (#HY-15763), sorafenib (#HY-10201), sulfasalazine (#HY-14655), DON (#HY-108357), RSL3 (#HY-100218A), L-Buthionine-(S,R)-sulfoximine (BSO, #HY-106376A), ferrostatin-1 (#HY-100579), Z-VADFMK (#HY-16658), AICAR (#HY-13417), BafA1 (#HY-100558), N-Acetylcysteine (#HY-B0215), and Necrosulfonamide (#HY-100573) were purchased from MedChemExpress (USA). Compound C (#ab120843) was purchased from Abcam. Deferoxamine mesylate (#D9533) was purchased from Sigma-Aldrich.

Complete Freund’s adjuvant (CFA) (#F5881) was purchased from Sigma-Aldrich. AICAR (#9944) was obtained from Cell Signaling Technology, and Compound C (# ab120843) was purchased from Abcam. Recombinant mouse IL-1ra/IL-1F3 (# 480-RM-050) was purchased from R&D systems.

Antibodies against GAPDH (sc-25778) were purchased from Santa Cruz Biotechnology. Antibodies against phospho-ACC (Ser⁷⁹) (CST3661), phospho-AMPKα (Thr¹⁷²) (CST2535) and total AMPK (CST2532) were purchased from Cell Signaling Technology. Bis-Tris 4–12% polyacrylamide gels (345–0123), MES running buffer (161–0789) and horseradish peroxidase secondary antibody conjugate (170–6515) were obtained from Bio-Rad. Polyvinylidene Fluoride (PVDF) Immobilon-P membrane (IPVH00010) was manufactured by Merck Millipore. Protein molecular weight marker (RPN800E) was purchased from GE Healthcare Life Sciences, while enhanced chemiluminescence (ECL) reagent (32209) and BCA protein assay were purchased from Life Technologies. Metformin was obtained from Calbiochem (Merck Millipore), 2-deoxy-D-glucose from Sigma-Aldrich and Compound C (Dorsomorphin) and A-769662 from Abcam. All other reagents, unless otherwise specified, were purchased from Sigma-Aldrich or Merck Millipore.