Fig 2: Effects of Kv1.5 on intracellular K+ concentration, ASC oligomerization and speck formation in J774.1 cells. Intracellular K+ concentrations of LPS-primed cells stimulated with MSU in either the presence or absence of DPO-1 (1 μM) or the selective Kv1.3 channel blocker PAP-1 (50 nM) (a), and those with the introduction of a siRNA against Kv1.5 or Kv1.3 or a scramble siRNA 24 h before the LPS and MSU treatments (b) (n = 5–11). *p < 0.01, †p < 0.05; NS: not statistically significant. c The LPS-primed and subsequently stimulated with MSU cells were incubated with OPTI-MEM containing different K+ concentrations (0–60 mM) overnight (n = 5). Shown are the representative blot. *p < 0.01 vs. LPS + MSU (+) & K+ 0 mM. d IBs of disuccinimidyl suberate (DSS)-crosslinked ASC oligomers in lysates from untreated cells and LPS-primed cells treated with DPO-1 (1 μM) or vehicle 30 min before MSU stimulation (n = 5). e Representative immunofluorescence images of ASC speck formation in LPS-primed MSU-stimulated cells with and without DPO-1 (1 μM) treatment. Arrows denote ASC specks. Averaged numbers of specked cells (% total) are shown by the bar graph (n = 5–6 fields). *p < 0.01

Fig 3: MARK4 deficiency affects MTOC and speck nucleation.(a) Differentiated THP-1 cells were stimulated with nigericin (10 μΜ for 1.5 h) or left untreated (control). Endogeneous levels of NLRP3 and MARK4 were co-stained with γ-tubulin. (b,c) HEK293T cells were co-overexpressed with GFP-MARK4, Cherry-NLRP3 and ASC-Flag (indicated as MARK4 GFP o.e.); or co-overexpressed with MARK4 shRNA (shown by green GFP), Cherry-NLRP3 and ASC-Flag (indicated as MARK4 shRNA). co-overexpression with MARK4-GFP drove NLRP3 to MTOC, and knock down of MARK4 by shRNA (indicated by GFP) led to a dilated ring structure of NLRP3. Quantification of speck size was shown. Scale bar, 40 μm. Mean±s.e.m. for all the cells taken from five different views at × 20 magnification for each group. Comparisons of the two different groups were analysed by unpaired t-test. ****P<0.0001 was considered as statistically significant (b). Mitochondria distribution was shown after speck formation (c). See also Supplementary Movie 6. (d) In differentiated THP-1 cells, upon nigericin stimulation (10 μΜ for 1.5 h), NLRP3 was translocated to MTOC, indicated by γ-tubulin. NLRP3 remained in cytoplasm distribution in MARK4 shRNA cell. Experiments were repeated at least three times. Scale bar, 10 μm.

Fig 4: The anti-aging protein klotho mitigates the NLRP3 inflammasome activation mediated by IL-1β in HUVEC. The expression of NLRP3 inflammasome components (A) NLRP3 and (B) pro-IL-1β was determined by Western blot in total lysates from HUVEC stimulated for 18-24 h with IL-1β (2.5 ng/ml) alone or in presence of r-klotho (1 nM). Representative blots are shown on top of the corresponding graphs, where β-Actin was employed as loading control (n= 6-8; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1β-treated cells, by two-way ANOVA and Tukey post-hoc test). (C) The NLRP3 inflammasome activation was determined by the detection of ASC specks by indirect immunofluorescence. The percentage of ASC specks per culture field was quantified under a fluorescence microscope (n= 3-4; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1β-treated cultures levels by two-way ANOVA and Tukey post-hoc test). (D) Confocal representative photographs (630X magnification) where white arrowheads mark the location of toroidal-shaped ASC specks and specific antibody against ASC (red) was used, while cell nuclei were counterstained with DAPI (blue). (E) IL-1β levels were measured in total cell lysates by Western blot, and also (F) detected in the supernatants of treated cells by ELISA (n= 4-5; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1β -treated cells, by two-way ANOVA and Tukey post-hoc test). All data are shown as mean ± SEM and expressed as percentage of IL-1β-induced levels.

Fig 5: IL-1β promotes priming and activation of the NLRP3 inflammasome in HUVEC. The expression of the NLRP3 inflammasome components (A) NLRP3 and (B) pro-IL-1β was determined by Western blot in total lysates from HUVEC stimulated for 18-24 h with IL-1β. Representative blots are shown on top of the corresponding graphs, where β-Actin was employed as loading control (n= 4-5; *p<0.05 vs. untreated cells by unpaired t-test). (C) The NLRP3 inflammasome activation was determined by the observation of ASC specks by indirect immunofluorescence after treatment with IL-1β alone or in presence of MCC 950 (1 µM). The percentage of ASC specks per cell culture field was quantified under a fluorescence microscope (n= 3-4; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1β-treated cells, by two-way ANOVA and Tukey post-hoc test). (D) Confocal representative photographs (630X magnification) where white arrowheads show the location of toroidal-shaped ASC specks as detected by a specific antibody against ASC (red), while cell nuclei were counterstained with DAPI (blue). (E) IL-1β, the final product of the NLRP3 inflammasome activation was measured in the supernatant of treated cells by ELISA (n= 4-5; *p<0.05 vs. untreated cells; #p<0.05 vs. IL-1β-treated cultures levels by two-way ANOVA and Tukey post-hoc test). HUVEC were treated with 2.5 ng/ml IL-1β in all experiments. All data are shown as mean ± SEM and expressed as percentage of IL-1β-induced levels.