Interleukin 6 (il 6)

MI-2 and mepazine (chemical structure shown in Figure 1A, synthetic compounds provided by Eternity Bioscience Inc. NJ, USA) was dissolved at a concentration of 30 mM in 100% DMSO as a stock solution, stored at −20°C, and diluted with medium before each experiment. The final DMSO concentration did not exceed 0.1% throughout the study (all the control groups are composed of 0.1% DMSO). Cyclosporine A (CsA), phorbol myristate acetate (PMA), lipopolysaccharide (LPS) and adenosine triphosphate (ATP) were purchased from Sigma-Aldrich (St. Louis, MO). Dextran sulfate sodium (DSS, 36–50 kDa) was bought from MP Biomedicals (Aurora, OH). Myeloperoxidase (MPO) activity assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). RPMI-1640, FBS, Alexa Fluor 546 Donkey Anti-Rabbit IgG and Alexa Fluor^® 488 Donkey Anti-Mouse IgG (H+L) were purchased from Life technology (Carlsbad, CA). Anti-phospho-IκBα, anti-phospho-IKKα/β, were purchased from Cell Signaling Technology (Beverly, MA). Anti-NLRP3, anti-phospho-p65 and anti-CASP1 were purchased from Epitomics (Burlingame, CA). Anti-ASC and anti-COX2 were purchased from Santa Cruz (Santa Cruz, CA). ELISA kits for murine TNF, IL-1β, IL-6, IFN-γ and human IL-1β were purchased from Dakewe Biotech Co. Ltd (Beijing, China). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Mouse IL-1β (R&D Systems, Minneapolis, MN, United States), IL-1β, TNF-α, and IL-6 (Dakewe, Beijing, China), according to the manufacturer’s instructions, were used to measure the cell culture supernatants and mouse serum, respectively.

Cytokine production of treated cells was measured by ELISA using the corresponding ELISA kits according to the manufacturers’ protocols. IL-6 (DKW1210602, Dakewe Biotech, China); TNF-α (DKW1217202 Dakewe Biotech, China); IL-10 (CME0016, 4A Biotech, China); TGF-β (DKW1217102 Dakewe Biotech, China). For NO measurement, culture supernatants were harvested after stimulation of macrophages with EST12 protein for indicated time. The release NO level was determined using the NO assay kit (Beyotime, Shanghai, China). Supernatants were added to an equal volume of Griess reagent in duplicate on a 96-well plate and incubated at room temperature for 15 min. Absorbance (540 nm) was measured and NO concentrations were estimated using a standard NO curve. The experiments were performed in triplicate.

GPR50 knockout 3T3‐L1 or control cells were incubated with 16.7 mm glucose or 0.25 mm palmitate for 48 h as described above. The supernatants were collected and centrifuged at 1509 g for 5 min. The concentrations of IL‐6 (Dakewe, Shenzhen, China), MCP‐1 (Sino Biological, Beijing, China), and IL‐1β (R&D Systems, Minneapolis, MN, USA) were assayed with ELISA kits according to the manufacturers' instructions. Quantitative data are presented as average concentrations in pg·mL⁻¹.