Fig 1: Pgam5 is required for microglia-induced Il-1β secretion and Asc oligomerization.NO production was detected by the (A) Griess reaction and cell viability was measured by (B) CCK8 assay. (A) *p < 0.001 compared to the control group and #p < 0.001 compared to the LPS group, by two-way ANOVA followed by Tukey’s post hoc multiple comparison test. C Primary microglia were primed with LPS for 4 h prior to stimulation with ATP for 30 min, and Il-1 β secretion was determined by ELISA. D Dynamically monitor Il-1β level in LPS-primed microglia treated with ATP for the indicated amount of time. C, D *p < 0.001 compared to the NC-sh group and #p < 0.001 compared to the Pgam5-sh group, by two-way ANOVA followed by Tukey’s post hoc multiple comparison test. (E) Whole-cell extracts were subjected to Western blot analysis. Microglia were stimulated with LPS + ATP and the mRNA of (F) cytokineand (G) pyroptosis-related genes were determined by qRT-PCR. *p < 0.001 compared to the WT group by one-way ANOVA followed by Tukey’s post hoc multiple comparison test. Data shown are mean ± SEM (n = 4). Pgam5-shRNA and caspase8-shRNA microglia were stimulated with LPS + ATP. H Results of immunoblotting with antibodies to the indicated proteins and immunoprecipitated with anti-Asc. I The detergent-insoluble fraction was subjected to chemical cross-linking, and monomers, dimers, and oligomers of Asc and Pgam5 were measured. J Immunofluorescence analysis of Asc specks (arrows) in microglia. After treatments, cells were fixed and stained for Asc (Red). Nuclear staining with DAPI (Blue). n = 4.