FIGURE 6.

Koumine (KM) blocks NLRP3 inflammasome assembly in BMDMs. BMDMs were pretreated with KM (200 μM) for 1 h and then incubated with LPS (300 ng/ml) for 3 h and ATP (5 mM) for 1 h. (A) Representative immunofluorescence images of ASC speck formation are shown. The white arrows indicate ASC specks (green), and the nuclei (blue) were stained with Hoechst 33324. Scale bars, 20 μm. (B) Quantification of cells with ASC specks in six random images. (C) ASC oligomerization in Triton-X100-insoluble pellets cross-linked with DSS was analyzed by western blotting. (D) The interaction between NLRP3 (red) and ASC (green) in BMDMs were assayed by immunofluorescence. Scale bars, 25 μm. (E,F) Cell lysates from cultured cells were analyzed by immunoprecipitation using an antibody against ASC and then analyzed by immunoblotting, and IgG was used as the isotype control. Mean ± SD of three independent experiments are shown. #p < 0.05 vs. LPS group, *p < 0.05 vs. LPS plus ATP group.