Extended Data Fig. 9: K⁺ efflux-independent stimuli also induced TGN dispersion and PtdIns4P-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 with imiquimod or CL097 (45 μg ml⁻¹) 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 owing to the partial separation of PtdIns4P and TGN38. Cells with NLRP3 puncta on dTGN were quantified from 100 cells (n = 3, mean ± s.d.; two-sided t-test). c, Neither imiquimod- nor CL097-induced NLRP3 puncta were co-localized with mitochondria in ASC-deficient BMDMs. Cells were primed with LPS (50 ng ml⁻¹) for 3 h and incubated with imiquimod or CL097 (45 μg ml⁻¹) for 60 min, before immunostaining for endogenous NLRP3 and TOM20 (mitochondrial marker). d, High extracellular KCl had no effect on imiquimod- or CL097-induced NLRP3 aggregation on dTGN. HeLa cells expressing NLRP3–GFP cells were treated with imiquimod or CL097 (45 μg m⁻¹) in the presence of KCl (0 or 30 mM) for 80 min before imaging. Results were analysed as in b. n.s., not significant (α = 0.01).