Fig. 6

25-HC induces inflammasome activation. ASC specks in microglia treated with LPS (10 ng/ml) without or with 25-HC (10 μg/ml) were stained by ASC antibody (green) and DAPI for nuclei (blue) (a). Quantification of ASC specks in microglia treated with medium alone (ctl), medium-containing LPS (10 ng/ml), 25-HC (10μg/ml), or LPS (10 ng/ml) plus 25-HC (10 μg/ml) (b). WT or TLR4 deficient microglia were treated with LPS (10 ng/ml) and 25-HC (10 μg/ml) for 24 h followed by ASC antibody staining (green) and DAPI (blue) (c) or ELISA measurements of secreted IL1β (d). Inhibition of caspase 1 by VX765 (e) or a high concentration of potassium (50 mM) (f) in the medium prevents 25-HC-dependent IL-1β production in microglia treated with 10 ng/ml LPS for 24 h. Statistical analyses were determined by ordinary two-way ANOVA with Tukey multiple comparisons test in c, e and f or Student t test in d. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. The data shown are representative of three or more independent experiments