Figure 5. Andrographolide inhibits CASP1 activation and IL1B maturation by interrupting the formation of the NLRP3 inflammasome in vitro. THP-1 (pretreated with 500 nM PMA for 3 h) or BMDM cells were cultured with 100 ng/ml LPS for 3 h, then treated with Andro (3, 10, or 30 μM) for 1 h, followed by 1 h incubation with 5 mM ATP. (A) IL1B levels in the supernatant fraction were analyzed by ELISA. Data are mean ± SEM of 3 different experiments. *P < 0.05, **P < 0.01 vs. LPS+ATP group. (B) Protein levels of pro-IL1B, IL1B p17, pro-CASP1, cleaved CASP1, PYCARD, and NRLP3 were determined by western blotting. Data shown are representative of 3 experiments. (C) CASP1 activity was measured. Data are mean ± SEM of 3 different experiments. *P < 0.05, **P < 0.01 vs. LPS+ATP group. (D) LPS-primed THP-1 cells were treated with 30 μM Andro for 1 h, followed by 2, 5, or 15 min incubation with 5 mM ATP. In the other experiment, LPS-primed THP-1 cells were treated with Andro (3, 10, or 30 μM) for 1 h, followed by 5 min incubation with 5 mM ATP. Proteins were isolated and immunoprecipitated with an antibody against PYCARD. Data shown are representative of 3 experiments. (E) LPS-primed BMDM cells were treated with 30 μM Andro for 1 h, followed by treatment with 5 mM ATP for 15 min. Cells were analyzed by immunofluorescence cytochemistry. Scale bar: 10 μm. Data shown are representative of 3 experiments. Andro, andrographolide.