Cells culture and treatments
Human monocyte cell line THP-1 was obtained from the Cell Bank of the Chinese Academic of Sciences (Shanghai, China). THP-1 cells were cultured in 1640 supplemented with 2-mercaptoethanol (0.5mM), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% FBS in an atmosphere of 5.0% CO2 at 37 °C. THP-1 cells can be induced to differentiate into macrophages by 0.5 mM PMA for 3h. Bone marrow-derived macrophages (BMDMs) were cultured in a complete DMEM medium supplemented with 10% FBS and 30ng/ml M-CSF (Cui et al., 2020). THP-1 cells and BMDMs were treated with LPS (1µg/ml; 6 h) in the absence or presence of AB4. In order to activate NLRP3 inflammasome, BMDMs and THP-1 cells were first treated by LPS (1µg/ml; 6 h), and cells were further co-treated with ATP (5 mM; 30 min), or nigericin (10 µM; 30 min), respectively. In some experiments in this paper, cells were first treated with the NF-κB inhibitor JSH-23 (25 µM; 10 h; #B1645, APExBIO Technology LLC, USA), the AKT inhibitor MK220 (20 µM; 10 h; #SF2712, Beyotine, Shanghai, China), the AKT agonist SC79 (20 µM; 12h; #SML0749, Sigma-Aldrich, Louis, USA), cells were further co-treated with LPS (1µg/ml; 6 h). Cell lysates were extracted for qPCR or Western blot, and supernatants were collected for ELISA to detect the release of related cytokines.