Hy 10355

The mouse peritoneal macrophages (MPMs) extracted from C57BL/6 female mice and MH-S cells and were cultured routinely in Roswell Park Memorial Institute (RPMI) medium (HyClone) supplemented with 10% fetal bovine serum (FBS), l-glutamine, and penicillin. Immortalized bone marrow-derived macrophage (iBMDM) cells and Raw264.7 cells were cultured routinely in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone) supplemented with 10% FBS, l-glutamine, and penicillin. All cells were cultured in antibiotic-free medium before PA stimulation or inhibitor pretreatment. The cells were pretreated with VX-765 (HY-13205, Med Chem Express), Nig (HY-13205, Med Chem Express), IL-1β (HY-P7073, Med Chem Express), IL-1β receptor inhibitor (TLR-1, HY-W011400, Med Chem Express), AKT inhibitor VIII (HY-10355, Med Chem Express), or IFN-β (CAT 300-02BC, PeproTech) for the indicated times before stimulation with PA, as indicated; multiplicity of infection (MOI) values are given for the different times.

The human HCC cell lines (Huh7, HepG2) were obtained from the cell bank of the Type Culture Collection of the Chinese Academy of Sciences. HEK-293T and HCC cell lines (SM-386, YY-8103, MHCC97-H) were obtained from ATCC cell bank, and routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco). L02 cells were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co. Ltd., and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% FBS. All cells were maintained in a humidified 5% CO₂ atmosphere at 37 °C, and tested for mycoplasma contamination every month. FCN3-overexpressed MHCC97-H, YY-8103 and control cells were treated with T0901317 (HY-10626, MedChemExpress), Erastin (HY-15736, MedChemExpress), FINO2 (HY-129457, MedChemExpress), AKTi-1/2 (HY-10355, MedChemExpress), Ferrostatin-1 (SML0583, Sigma-Aldrich), N-acetyl-L-cysteine (A9165, Sigma-Aldrich), Z-VAD-fmk (FMK001, R&D Systems), and Necrostatin-1 (HY-15760, MedChemExpress) for indicated time.