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Fig. 5 | Journal of Neuroinflammation

Fig. 5

From: Directly targeting ASC by lonidamine alleviates inflammasome-driven diseases

Fig. 5

LND blocks the formation of ASC specks. A–C LPS-primed BMDMs were treated with LND (0.2 mM) for 30 min before 30 min of ATP treatment. Western blot analysis of NLRP3 protein in the cell lysates that immunoprecipitated with anti-NEK7 antibodies to evaluate the NLRP3-NEK7 interaction (A). Western blot analysis of ASC protein in the cell lysates that immunoprecipitated with anti-NLRP3 antibodies to evaluate the ASC-NLRP3 interaction (B). Western blot analysis of caspase-1 protein in the cell lysates that immunoprecipitated with anti-ASC antibodies to evaluate the pro-cas-1-ASC interaction (C). D and E LPS-primed BMDMs were treated with LND (0.2 mM) for 30 min before 30 min of ATP treatment. Western blot (D) and quantitative analysis (E) of crosslinked ASC in the NP-40–insoluble pellet with anti-ASC antibodies. F Immunofluorescence analysis of ASC speck formation in LPS-primed BMDMs treated with various doses of LND. Arrowheads depict ASC specks. Scale bar = 10 μm (G) The percentage of ASC speck in (F). H Immunofluorescence analysis of ASC in the spinal cords of EAE mice. White arrows depict ASC specks. Scale bar = 25 μm. I and J LPS-primed BMDMs were treated for 0.5 h with LND (0.2 mM) and then stimulated with inflammasome activators (ATP (5 mM, 30 min), poly(dA:dT) (1 μg/ml, 8 h), MDP (200 ng/ml, 8 h) or flagellin (200 ng/ml, 8 h)), respectively. Western blot analysis of cleaved caspase-1 (p20) in the supernatants (SN) of BMDMs and pro-caspase-1 in the lysates (I). ELISA analysis of IL-1β in the supernatants of BMDMs (J). The data are expressed as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. The data were analyzed by one-way ANOVA and Tukey’s test

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