Extended Data Fig. 2: NLRP3 aggregates on stimulus-triggered dTGN.

a, Nigericin treatment induced formation of giant vesicles in the perinuclear region. HeLa cells expressing NLRP3–GFP treated with nigericin (10 μM) for 80 min were examined with phase-contrast microscopy. Mag, magnification. Cells with giant vesicle formation was quantified from 100 cells (n = 3, mean ± s.d., two-sided t-test). N.D., not detectable. b, Ultrastructural analysis of nigericin-induced dispersed TGN (dTGN) vesicles. HeLa cells treated as in a were examined by transmission electron microscopy. Blue arrowheads indicate Golgi stacks under resting conditions and red arrowheads indicate nigericin-induced dTGN vesicles. Representative images from two independent experiments (more than 30 cells were examined for each condition in each experiment) are shown. c, Nigericin triggered the formation of dTGN, on which NLRP3 aggregated. HeLa cells stably expressing the indicated protein were stimulated as in a before immunostaining for the TGN markers TGN38 and GOLGA4. d, e, cis and medial Golgi remained intact after nigericin treatment. Cells treated as in a were immunostained for GM130 (cis-Golgi marker) or giantin (cis- and medial-Golgi marker). f, NLRP3 aggregated on dispersed TGN38-positive vesicles, some of which were also EEA1-positive. HeLa cells stably expressing NLRP3–GFP and EEA1–HA were treated as in a before immunostaining for TGN38 and HA. g, ATP stimulation led to NLRP3 aggregation on dTGN. HeLa cells stably expressing NLRP3–GFP and P2X₇–HA were treated with ATP (5 mM) for 80 min before imaging. P2X₇ is a purinergic receptor that is essential for ATP-mediated NLRP3 inflammasome activation. h, Stimulation with gramicidin led to NLRP3 aggregation on dTGN. HeLa cells were treated with gramicidin (5 μM) for 80 min before imaging. i, DNA stimulation does not cause TGN dispersion or AIM2 recruitment to TGN. HeLa cells stably expressing AIM2–Flag were mock-transfected, transfected with poly(dA:dT) (1.5 μg ml⁻¹) for 3 h or incubated with nigericin (10 μM) for 80 min before immunostaining with antibodies against Flag (AIM2–Flag) or TGN38.