Extended Data Fig. 5: The polybasic region mediates dTGN recruitment and activation of NLRP3.

a, NLRP3 is not required for dTGN formation in response to nigericin in HeLa cells. HeLa cells (without NLRP3 expression) were treated with nigericin (10 μM) for 80 min and immunostained for TGN38. Cell borders are outlined with dashed lines. b, ATP induced dTGN formation in a manner dependent on P2X₇ but not NLRP3. HeLa cells or HeLa cells stably expressing P2X₇–HA were incubated with ATP (5 mM) for 80 min before immunostaining for TGN38. c, Constitutively active mutants of NLRP3 can bypass the TGN-recruitment step by spontaneously forming aggregates in the cytosol. Cells stably expressing the indicated proteins were untreated or treated with nigericin (10 μM) for 80 min before immunostaining for TGN38. d, Immunoblots of cells used in Fig. 3c. e, The KKKK motif is critical for nigericin-induced NLRP3 activation. Cells stably expressing the indicated proteins were untreated or treated with nigericin (10 μM) for 60 min before being tested in the in vitro NLRP3 activation assay. f, Mutations in the KKKK motif do not compromise the ability of constitutively active NLRP3 to activate caspase-1. Cells stably expressing the indicated proteins were examined by fluorescence microscopy (top) and the in vitro NLRP3 activity assay (bottom) without stimulation. LP, NLRP3(L351P).