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. Author manuscript; available in PMC: 2022 Aug 25.
Published in final edited form as: Nature. 2018 Nov 28;564(7734):71–76. doi: 10.1038/s41586-018-0761-3

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

Extended Data Figure 1.

a, Endogenous NLRP3, ASC and caspase-1 are not detectable in HEK293T. Extracts from HEK293T cell lines stably expressing the indicated proteins were examined by immunoblotting. *, a nonspecific band. b, Reconstitution of NLRP3 inflammasome pathway in HEK293T. Cells stably expressing murine NLRP3, ASC and caspase-1 were treated with nigericin (Nig) (10 μM) for 60 minutes followed by immunoblotting. c, Highly purified NLRP3 showed signal-dependent activity in the in vitro assay. NLRP3-GFP was purified through fractionation and immunoprecipitation as detailed in Methods, before the purity and activity were examined by silver staining (left panel) and the in vitro assay (right panel) respectively. d, The in vitro assay detects activation of endogenous NLRP3. RAW 264.7 was treated with LPS (50 ng/mL) for 3 hours and stimulated with nigericin (10 μM) for 60 minutes before cell extracts were collected for the in vitro NLRP3 activity assay. e, Human NLRP3 also formed various puncta in response to nigericin. HeLa cells stably expressing human Flag-NLRP3 were treated with nigericin (10 μM) for 80 minutes before immunostaining with a Flag antibody. f, NLRP3 formed multiple puncta in the absence of ASC but 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 panel: nigericin (10 μM, 60 minutes)-induced NLRP3 puncta remained in the cells after saponin treatment. Nu, Nucleus. Right panel: 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 of that in Lane 6 based on quantification in imageJ (normalization by NLRP3 bands 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.