Fig 2: Evodiamine-mediated enhancement of ATP- or nigericin-induced NLRP3 inflammasome activation was attenuated by blocking acetylation of α-tubulin. (A) LPS-primed BMDMs were pre-treated with resveratrol (5 μM), NAD+ (10 μM) or ciliobrevin A (30 μM) for 30 min, and then incubated with evodiamine (5 μM) for 1 h, or followed by stimulation with nigericin (5 μM) for 1 h. Representative immunofluorescence images showing acetylated α-tubulin (red) subcellular distribution. Nuclei (blue) were revealed by Hoechst 33342. The images for acetylated α-tubulin and nuclei were captured, respectively, and merged together. Scale bars, 10 μm. (B) LPS-primed BMDMs were pre-treated with resveratrol (5 μM) for 30 min, and then incubated with indicated dose of evodiamine for 1 h, followed by stimulation with ATP (2 mM) for 30 min. Representative images showing ASC (green) subcellular distribution. The images for ASC and nuclei were captured, respectively, and merged together. Yellow arrows indicate ASC specks and the enlarged inset showing cells with an ASC speck. Scale bars, 20 μm. (C) Percentages of cells with an ASC speck relative to the total number of cells from 5 random fields (one field per well) each containing ∼200 cells. Data are shown as mean ± SD (n = 5). ∗P < 0.05, ∗∗∗P < 0.001, two-tailed Student’s t-test. (D) BMDMs were treated as in Figure 4A. The levels of soluble IL-1β in culture supernatants were measured by cytometric bead array (CBA) assay. The experiments were performed three times independently. Data are shown as mean ± SD (n = 5). One-way ANOVA: P < 0.0001; Tukey post hoc test: ∗∗P < 0.01, ∗∗∗P < 0.001; RSV, resveratrol; EVO, evodiamine; Nig, nigericin.

Fig 3: Evodiamine specifically promoted NLRP3 inflammasome activation in macrophages. (A) The chemical strcuture of evodiamine. (B–D) J774A.1 cells (B) and bone marrow-derived macrophages (BMDMs) (C,D) were treated as described in Section “Materials and Methods.” Western blotting was used to assess the expression levels of indicated proteins in the cell lysates and culture supernatants, respectively. Actin was used as a loading control for cell lysates. (E) Human THP-1 macrophages were induced by PMA (500 nM) treatment for 16 h. The cells were then primed with LPS (1 μg/ml) for 4 h, and pre-treated with series concentrations of evodiamine for 1 h, followed by incubation with nigericin (20 μM) for 1 h in the absence of LPS. The levels of soluble IL-1β in the culture supernatants were detected by cytometric bead array (CBA) assay. Data are shown as mean ± SD (n = 5). One-way analysis of variance (ANOVA): P < 0.0001; Tukey’s post hoc test: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (F) BMDMs were treated as in (C). Representative immunofluorescence images showing ASC (red) subcellular distribution. Nuclei (blue) were revealed by Hoechst 33342. Yellow arrows indicate ASC specks and the enlarged inset showing cells with an ASC speck. Scale bars, 20 μm. (G) Percentages of cells with an ASC speck relative to the total number of cells from 5 random fields (one field per well) each containing ∼50 cells. Data are shown as mean ± SD (n = 5). One-way ANOVA: P < 0.0001; Tukey’s post hoc test: ∗∗∗P < 0.001. (H) Western blot analysis for ASC in Triton-X 100 insoluble cytosolic pellets cross-linked with disuccinimidyl suberate. (I) J774A.1 cells were primed with Pam3CSK4 (1 μg/ml) for 4 h, then pre-treated with evodiamine 1 h, and followed by transfection with 2 μg/ml Poly(dA:dT), 0.5 μg/ml flagellin or 2.5 μg/ml LPS for 16 h, respectively. Soluble IL-1β levels in the culture supernatants was quantified by CBA assay. Data are shown as mean ± SD (n = 5). The experiments were performed three times independently. ∗∗∗P < 0.001, ns, not significant, by two-tailed Student’s t-test. EVO, evodiamine; Pam, Pam3CSK4.