Extended Data Fig. 4: NLRP3 activity is strongly associated with dTGN but not mitochondria.

a, NLRP3 did not translocate to mitochondria upon stimulation in HeLa cells. HeLa cells expressing NLRP3–GFP were stimulated with nigericin (10 μM) or gramicidin (5 μM) for 80 min and were immunostained for TOM20 (mitochondrial marker). b, Neither nigericin- nor ATP-induced NLRP3 puncta were co-localized with mitochondria in ASC-deficient BMDMs. Cells were primed with LPS (50 ng ml⁻¹) for 3 h, followed by nigericin (10 μM) or ATP (5 mM) treatment for 60 min before immunostaining for endogenous NLRP3 and TOM20. c, NLRP3 activity in P100 (light membrane) fraction was strongly associated with dTGN but not mitochondria. P100 fraction collected from Fig. 1c was separated by sucrose gradient ultracentrifugation. Fractions were collected and tested for activity in the in vitro NLRP3 activity assay (top). TOM20 (mitochondrial marker) and GM130 (cis-Golgi marker) were not detectable on immunoblots even after prolonged exposure. d, dTGN-localized NLRP3 puncta can initiate aggregation of ASC-PYD. HeLa cells stably expressing the indicated proteins were incubated with nigericin (10 μM) for 80 min before imaging. ASC-PYD, residues 1–90 of mouse ASC. Right, top to bottom: reconstituted z-stack image of a representative nigericin-treated cell; nigericin-treated cell co-immunostained for TGN38 (pseudocoloured in magenta); cells with ASC-PYD filaments originating from dTGN-localized NLRP3 puncta were quantified from 100 cells (n = 3, mean ± s.d., two-sided t-test).