Rapamycin

Antibodies used in the study included rabbit anti-mouse caspase-1 p10 (sc-514) and anti-mouse ASC (sc-22514-R) (Santa Cruz, CA, United States); rabbit anti-mouse NLRP3 (Abcam, Cambridge, MA, United States); rabbit anti-mouse LC3B (MBL, Japan); and rabbit anti-mouse β-actin (Beyotime, Wuhan, Hubei, China); rabbit anti-rat TLR4 (Proteintech, Chicago, IL, United States); rabbit anti-mouse TRIF (Abcam, Cambridge, MA, United States); rabbit anti-mouse GAPDH (Proteintech, Chicago, IL, United States); goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody (ZSGB Biotechnology, Beijing, China). Lipopolysaccharides (LPS, E. coli L2630) and 3-MA (M9281) were from Sigma-Aldrich (St. Louis, MO, United States), and rapamycin was from Beyotime Biotechnology (Wuhan, Hubei, China). Caspase-1 inhibitor Z-YVAD-FMK was purchased from Biovision (San Francisco, CA, United States). The ELISA kit for mouse IL-1β (88-7013) was purchased from eBioscience Technology (San Diego, CA, United States). Reagents and apparatus used in immunoblotting assays were obtained from Bio-Rad (Hercules, CA, United States).

Hy was purchased from Shanghai Shifeng Technology Co., Ltd. (Shanghai, China). RNAiso Plus reagent was purchased from Takara Co. (Takara, Otsu, Shiga, Japan). Cell culture supplies were purchased from Costar (Corning Inc., Cypress, CA, USA). MCDB 131, lipopolysaccharide (LPS), GW9662 and pioglitazone were purchased from Sigma Chemical Co. (St Louis, MO, USA). Antibody against glyceraldehyde phosphate dehydrogenase (GAPDH), total or phosphorylated mTOR and goat anti-rabbit IgG H&L (Alexa Fluor^® 488) were purchased from Abcam (Cambridge, MA, USA). Antibodies against total or phosphorylated Akt were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against toll-like receptor 4 (TLR4), peroxisome proliferator-activated receptor γ (PPAR-γ) and HRP-labeled goat anti-rabbit secondary antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd. (Chinese branch, Wuhan, China). LY294002, rapamycin, and DAPI were obtained from Beyotime Biotechnology Research Institute (Shanghai, China). The specific primers, siRNA sequences targeted TLR4 and negative siRNA sequences were synthesized by SangonBiotech (Shanghai, China) Co., Ltd. (Shanghai, China). The concentration of inhibitors used in the present study was determined according to previous studies and our preliminary studies. All experiments were conformed to the Medical Ethics Committee of Jiangnan University.

To estimate the role of TREM-1 in LPS-activated macrophages, we treated cells with LR12 (25 μg/mL) 30 min before LPS (1 ng/mL) stimulation. To study TREM-1-mediated activation, we pre-coated 6-well plates (2×10⁶ cells/well), 12-well plates (1×10⁶ cells/well), or 24-well plates (0.5×10⁶ cells/well) with agonist anti-TREM-1 mAb (10 μg/mL, Mab1187, R&D Systems, USA) overnight at 37 ℃ and washed twice with PBS. Then purified macrophages were subjected to additional stimuli, including glycolysis inhibitor (2-DG, 5 mM, Sigma-Aldrich), mTOR inhibitor (Rapamycin, 100 nM, Beyotime, Jiangsu, China), PI3K inhibitor (LY294002, 25 μM, Beyotime), HIF-1α inhibitor (PX-478, 25 μM, Medchem Express, USA), scavenger of ROS (NAC, 500 μM, Beyotime), or DRP1 inhibitor (Mdivi-1, 100 nM, Medchem Express). The macrophages were cultured for 6 or 24 h and harvested for gene or protein detection.

Dulbecco’s modified Eagle’s medium/F12 (1∶1, DMEM/F12,) and B27 were from Gibco (Grand Island, NY, USA). Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), trypsin, and dimethyl sulfoxide (DMSO) were purchased from Sigma (St Louis, MO, USA). IGF-1, PD98059, and LY294002 were from Invitrogen (Carlsbad, CA, USA). Both PD98059 and LY294002 were dissolved in DMSO and stored at 50 mM. Rapamycin was from Beyotime (Nantong, CHN). AG1024 was form Selleck (Housten, TX, US). Other reagents are described below.

The cell lines SKOV3, ES2, and HEK293T were obtained from ATCC (Manassas, VA, USA). The A2780 cell line was acquired from ECACC (Salisbury, UK). A2780 and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Biological Industries (BI), Israel), ES2 cells were cultured in McCoy’s 5A medium (BI), and SKOV3 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (BI). All media were supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and cells were cultured at 37 °C in a 5% CO₂ incubator. All cell lines were authenticated by genetic profiling using polymorphic short tandem repeat loci. The cells were treated with LY294002 (Beyotime, Shanghai, China) or rapamycin (Beyotime) at the indicated concentrations for 24 h, before subsequent analysis.