Figure 4

Figure 4

AP blocks NLRP3 inflammasome assembly by hindering ATPase activity of NLRP3. (a) HEK 293FT cells were transfected with NLRP3-Myc and treated with or without AP for 2 h. The interaction between NLRP3 and NEK7 was analyzed by immunoprecipitation and immunoblot. (b) The ATPase activity of NLRP3 with or without AP was measured by luminescence using the ADP-Glo™ Max assay. (c) LPS-primed THP-1 cells were treated with AP for 2 h and activated with nigericin (10 μM) for 30 min. The cell pellet was cross-linked by DSS (2.5 mM) for 30 min. ASC oligomerization was analyzed by immunoblot. (d,e) LPS-primed J774A.1 cells treated with AP for 2 h and activated with nigericin (10 μM) for 30 min. Representative immunofluorescence images of ASC speck formation (indicated by arrows) were taken by a confocal laser-scanning microscope (Carl Zeiss, LSM710, scale bar, 20 μm) (d). Graph representing the number of cells containing ASC specks in all treatments (e).