Extended Data Fig. 8: Binding to PtdIns4P on dTGN is essential for NLRP3 inflammasome activation.

a, b, The KKKK motif of NLRP3 can be functionally replaced with a PtdIns4P-binding domain of OSBP (OSBP-PH). a, Cells stably expressing the indicated proteins were treated with nigericin (10 μM) for 80 min before imaging. Cells with NLRP3 on dTGN were quantified from 100 cells (n = 3, mean ± s.d., two-sided t-test). 2RE, NLRP3(R107/108E). Note that NLRP3(ΔKKKK OSBP-PH) that constitutively localized on intact TGN without stimulation was not counted in the quantification. b, Replacement of the KKKK motif with OSBP-PH allowed NLRP3 to be constitutively localized on TGN. Cells were treated as in a before immunostaining for TGN38. c, Mutations of OSBP-PH domain that abrogate its binding to PtdIns4P also abolish its ability to functionally rescue NLRP3(ΔKKKK). Cells were treated as in a and cell extracts were examined by the in vitro NLRP3 activity assay. d, Immunoblots for primary NLRP3-deficient BMDMs rescued with Flag–NLRP3 (wild type or mutants), which were used for experiment in Figs. 5b, 6d. The cells were infected with lentivirus encoding the indicated proteins for 6 days before immunoblotting. e, Recruitment of NLRP3 to non-PtdIns4P-enriched regions of TGN is not sufficient to support its activation. Cells stably expressing the indicated proteins were treated as in a before examination by fluorescence microscopy (left; images for NLRP3(ΔKKKK OSBP-PH) are shown in a) and the in vitro NLRP3 assay (right). f, Extracellular KCl at 30 mM was sufficient to completely block nigericin-induced K⁺ efflux. Cells were treated with nigericin (10 μM) for 80 min in the presence of increasing concentrations of KCl, and cell extracts were collected for measurement of intracellular K⁺ concentration (shown as percentage change compared to untreated sample, mean ± s.d.). Representative results from two independent experiments (each containing two samples for each condition) are shown. g, Incubation in K⁺-free medium induced spontaneous K⁺ efflux. Cells were incubated in Hanks’ buffer containing 5 mM K⁺ for the first two conditions, or Hanks’ buffer in which K⁺ was replaced by Na⁺ for the third condition (K⁺-free). The second condition also contained nigericin (10 μM). After 80 min, the cell extracts were collected for intracellular K⁺ measurement using methods similar to f. h, K⁺ efflux alone is not sufficient to activate either wild-type NLRP3 or NLRP3(ΔKKKK OSBP-PH). Cells stably expressing the indicated proteins were treated as in g before testing with the in vitro NLRP3 activity assay. i, Incubation in K⁺-free medium induced spontaneous K⁺ efflux in primary wild-type BMDMs. Cells were primed with LPS (50 ng ml⁻¹) for 3 h, before treatment as in g for the indicated time period. Intracellular K⁺ concentrations were then measured and analysed as in f. j, K⁺ efflux alone is not sufficient to activate endogenous NLRP3 in primary wild-type BMDMs. Cells treated as in i were used for immunoblotting. Lys, lysate; sup, supernatant.