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. 2020 Oct 23;6(43):eaba4733. doi: 10.1126/sciadv.aba4733

Fig. 5. EST12 triggers macrophage pyroptosis and IL-1β secretion via NLRP3 and GSDMD.

Fig. 5

(A to C) WT, NLRP3−/−, and GSDMD−/− peritoneal macrophages treated with EST12 at the indicated doses (A) or for the indicated time periods (B and C) were analyzed for cytotoxicity by LDH assay (A and B) and IL-1β secretion by ELISA (C). Two-way ANOVA with Bonferroni’s multiple comparison test. (D) WT and NLRP3−/− peritoneal macrophages treated with EST12 were detected for GSDMD and caspase-1 activation by WB. (E) Peritoneal macrophages treated with EST12 and EST12-Y80A were detected for GSDMD activation by WB. (F to H) WT and caspase-1/11−/− peritoneal macrophages treated with EST12 were detected for LDH assay (F), secreted IL-1β by ELISA (G), and GSDMD activation by WB (H). Two-way ANOVA with Bonferroni’s multiple comparison test was used. (I and J) WT, NLRP3−/−, GSDMD−/−, and caspase-1/11−/− BMDMs infected with H37Rv and H37RvΔEST12 (I) or BCG and BCG-EST12 for 6 hours (J) were analyzed by LDH assay and ELISA. Unpaired t test versus H37Rv (I) or BCG (J). The data are expressed as the mean ± SEM of n = 3. NS (P > 0.05), **P < 0.01, and ***P < 0.001.