Extended Data Fig. 1: NLRP3 forms multiple puncta to activate the inflammasome pathway.

a, Endogenous NLRP3, ASC and caspase-1 are not detectable in HEK-293T cells. Extracts from HEK-293T cell lines stably expressing the indicated proteins were examined by immunoblotting. *, non-specific band. b, Reconstitution of NLRP3 inflammasome pathway in HEK-293T cells. Cells stably expressing mouse NLRP3, ASC and caspase-1 were treated with nigericin (Nig) (10 μM) for the indicated time, followed by immunoblotting. c, Highly purified NLRP3 showed signal-dependent activity in the in vitro assay. NLRP3–GFP was purified by fractionation and immunoprecipitation as detailed in Methods. Purity and activity were examined by silver staining (left) and the in vitro assay (right), respectively. d, The in vitro assay detects activation of endogenous NLRP3. RAW 264.7 cells were treated with LPS (50 ng ml⁻¹) for 3 h and stimulated with nigericin (10 μM) for 60 min before cell extracts were collected for the in vitro NLRP3 activity assay. e, Human NLRP3 also formed puncta in response to nigericin. HeLa cells stably expressing Flag–NLRP3 were treated with nigericin (10 μM) for 80 min before immunostaining with a Flag antibody. f, NLRP3 formed multiple puncta in the absence of ASC, and formed a large speck in the presence of ASC. Cells stably expressing the indicated proteins were treated as in e before imaging. g, NLRP3 puncta possessed high activity. Left, NLRP3 puncta induced by nigericin (10 μM, 60 min) remained in the cells after saponin treatment. Nu, nucleus. Right, cell extracts with or without saponin treatment were examined by the in vitro assay. The p10 level in lane 4 is approximately 6.5 fold that in lane 6 based on quantification in imageJ (normalization by NLRP3 band intensity). Only activator cell extracts were used for tubulin immunoblot. h, Constitutively active mutants of NLRP3 formed puncta without stimulation. Cells stably expressing the indicated proteins were imaged in the absence of stimulation.