Extended Data Figure 9. K⁺ efflux-independent stimuli also induced TGN dispersion and PI4P-dependent NLRP3 recruitment.

a, b, K⁺ efflux-independent stimuli also induced NLRP3 aggregation on dTGN through the KKKK motif. HeLa cells stably expressing the indicated proteins were treated −/+ imiquimod or CL097 (45 μg/mL) for 80 min before imaging. High magnification images are shown in the inset. Arrows indicate representative plasma-membrane-localized NLRP3 puncta, which were separated from TGN38-positive compartments due to the partial separation of PI4P and TGN38. The percentage of cells with NLRP3 puncta on dTGN was quantified from 100 cells (n = 3, mean ± SD; two-sided t test). N.D., not detectable. c, Neither imiquimod- nor CL097-induced NLRP3 puncta were colocalized with mitochondria in ASC-deficient BMDMs. Cells were primed with LPS (50 ng/mL) for 3 hours and incubated −/+ imiquimod or CL097 (45 μg/mL) for 60 min, before immunostained for endogenous NLRP3 and TOM20 (mitochondrial marker). d, High extracellular KCl had no significant effect on imiquimod- or CL097-induced NLRP3 aggregation on dTGN. HeLa NLRP3-GFP cells were treated −/+ imiquimod or CL097 (45 μg/mL) in the presence of KCl (0 or 30 mM) for 80 min before imaging. Results were analyzed as in (b). n.s., not significant (alpha = 0.01).