Extended Data Fig. 6: The polybasic region of NLRP3 functions through its positive charge.

a, Mutations of the KKKK motif to arginine do not affect nigericin-induced NLRP3 puncta formation. HeLa cells stably expressing the indicated proteins were treated with nigericin (10 μM) for 80 min before imaging. Cells with NLRP3 puncta were quantified from 100 cells (n = 3, mean ± s.d., two-sided t-test). n.s., not significant (α = 0.01). 2KR, NLRP3(K127R/K128R); 3KR, NLRP3(K127R/K128R/K129R); 4KR(K127R/K128R/K129R/K130R). b, Mutations of the KKKK motif to arginine do not impair nigericin-induced NLRP3 activation. Cells stably expressing the indicated proteins were treated with nigericin (10 μM) for 60 min before cell extracts were examined in the in vitro NLRP3 activity assay. c, The positive charge of the KKKK motif is important for gramicidin- and ATP-induced NLRP3 puncta formation. HeLa cells stably expressing the indicated proteins were treated with gramicidin (5 μM) (left) or ATP (5 mM) (right) for 80 min, before the percentage of cells with NLRP3 puncta was analysed as in a. d, The positive charge of the KKKK motif is essential for NLRP3 to polymerize ASC. Cells stably expressing the indicated proteins were treated with nigericin (10 μM) or gramicidin (5 μM) for 80 min before immunostaining for both NLRP3 and ASC. The percentage of cells with NLRP3–ASC specks was analysed as in a. e, Immunoblots of cells used in Fig. 3d. f, g, The second positively charged region of NLRP3 is also important for its aggregation on dTGN and activation in a manner dependent on the number of remaining positively charged residues. Cells stably expressing the indicated proteins were treated with nigericin (10 μM) before imaging (f) or examined by the in vitro NLRP3 activity assay (g).