Hy p7073

HT22 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone), supplemented with 10% fetal bovine serum (FBS) (Biological Industries) and 1% penicillin–streptomycin (Beyotime) in a 5% CO₂ incubator (Thermo Fisher Scientific) at 37°C. Plasmid transfection was performed when cells were approximately 70–80% confluent using Lipofectamine 2000 (Invitrogen). HT22 cells were treated with IL-1β for 48 h at a concentration of 10 ng/ml (MedChemExpress, HY-P7073). Cells were collected at the end of the treatment, and RNA or protein was extracted for further analysis. All experiments were repeated at least three times.

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.