Fig. 2.
Chloride efflux is sufficient to induce NLRP3-dependent ASC speck formation. (A, i) LPS-primed immortalized BMDMs stably expressing ASC-mCherry (ASC-mCherry iBMDMs) were incubated in a control (145 mM NaCl/5 mM KCl), Cl− free (145 mM NaGluconate/5 mM KGluconate), K+ free (150 mM NaCl), or K+ and Cl− free (150 mM NaGluconate) solution and ASC speck formation was measured in real time (n = 5). (A, ii) ASC speck formation was analyzed after a 3-h incubation in the respective solutions (n = 5). (A, iii) Representative images of ASC-mCherry iBMDM cells after a 3-h incubation in the respective solutions. (Scale bar, 50 µm, arrows denote ASC speck.) (B, i) LPS-primed ASC-mCherry iBMDMs were incubated in a control, Cl− free, K+ free, K+ and Cl− free, high K+ and normal Cl− (150 mM KCl), or high K+ and Cl− free (150 mM KGluconate) solution and ASC speck formation was measured in real time (n = 4). (B, ii) Speck formation was analyzed after a 3-h incubation in the respective solutions (n = 4). (C, i) LPS-primed WT and NLRP3 KO THP-1 macrophages were incubated in a control, Cl− free, K+ free, or K+ and Cl− free solution for 3 h and immunostained for ASC (n = 4). (C, ii) Representative images of WT and NLRP3 KO THP-1 macrophages incubated for 3 h in the respective solutions and immunostained for ASC. (Scale bar, 50 µm, arrows denote ASC speck.) All experiments were performed in the presence of Z-VAD-FMK (50 µM) to prevent pyroptosis and loss of ASC specks. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significantly different determined by a one-way ANOVA with Tukey’s (A and B) post hoc analysis, or two-way ANOVA with Sidak’s post hoc analysis (C, #P < 0.01 vs. WT in the respective solution). Values shown are the mean ± SEM.