Extended Data Fig. 3: K+ efflux was essential for ASC recruitment induced by multiple CDCs.
a–c, Representative immunofluorescence images (left) and quantification of TGN dispersion and NLRP3 recruitment (right) in HeLa cells stably expressing NLRP3-GFP incubated with 1.8 nM rALO (a), 14.4 nM rSLO (b), or 5.4 nM rPLY (c) for 80 min in the presence of KCl at the indicated concentrations. Scale bar, 10 μm. Areas containing TGN structures were measured with ImageJ (n = 40 cells per condition, mean ± s.d.; two-sided t-test; NS, not significant). NLRP3 recruitment was quantified from 100 cells (n = 3; N.D., not detectable). Representative from 3 independent experiments. d, Representative immunofluorescence images (left) and quantification of ASC speck formation (right) in HeLa cells stably expressing NLRP3-GFP and ASC incubated with 1.8 nM rALO, 14.4 nM rSLO or 5.4 nM rPLY for 60 min in the presence of KCl at the indicated concentrations. Scale bar, 25 μm. The percentage of cells with ASC speck formation was quantified from 100 cells (n = 3). Representative from 3 independent experiments. e, Model: CDCs represent a third type of NLRP3 stimuli. The canonical K+ efflux-dependent stimuli (for example, nigericin) does not require K+ efflux for TGN dispersion but requires K+ efflux for NLRP3 recruitment to the dispersed TGN. The K+ efflux-independent stimuli (for example, imiquimod) does not require K+ efflux for any step. CDCs do not require K+ efflux for either TGN dispersion or NLRP3 recruitment but require K+ efflux for ASC recruitment.
