Supplementary Figure 2: Excessive mitochondrial fission and dysfunction linked with inflammasome activation in microglia.

(a) Schematic of the experimental design using primary rat microglia treated with LPS (10 ng/ml) for 3 hours, followed by nigericin (1.2µM)-treatment in the presence/ absence of P110 (1 µM added 15 minutes prior to LPS) for 21 hours in serum and antibiotic free DMEM (top). Cellular levels of phospho-p44/42, phosphorylated NFκB and phosphorylated c-Jun N-terminal kinase (JNK) were determined by immunoblotting, quantified and presented as fold-change of control in rat primary microglia; (n=3). (b) Levels of Il-1β and caspase1-p20 were determined in cultured media of these primary microglia by immunoblotting. β-actin in the cells from the corresponding cultures was used as a cell loading control; (n = 2.) (c) Mitochondrial NLRP3 and ASC levels were determined by immunoblotting of mitochondrial fraction, quantified and presented as fold-change of control in these primary microglia. Data were evaluated by one-way ANOVA and Holm-Sidak’s multiple comparisons test for multiple testing between each treatment group; (n=5) All graphs represent mean ± s.d.