Fig. 1

Activation of the NLRP3 inflammasome by PAMPs in SIM-A9 microglial cells after cholesterol enrichment. Cells were treated with the CHO:MCD complex for 1 h. Inflammasome was induced by co-incubation with LPS and MDP (L + M, 10 μg/ml each for 16 h), LPS (10 μg/ml for 16 h) and ATP (5 mM for 1 h) (L + A), or MSU (150 μg/ml for 4 h). a Total cholesterol levels of cellular extracts. (n = 3 independent experiments). b Filipin staining. Representative images from 3 independent experiments. Scale bar: 100 μm. Filipin staining was quantified as the CTCF of the green channel (n = 12). c Western blot analysis of NLRP1, NLRP3 and IL-1β in cellular extracts. (pro-IL-1β: IL-1β pro-form). ACTB/actin β and ponceau S (PS) staining were used as loading controls. Optical density (O.D.) values of the bands representing the specific protein immunoreactivity were normalized to PS staining (n = 4). d Representative confocal images of oligomeric ASC from 3 independent experiments. Cells were transfected with a plasmid encoding an ASC:GFP fusion protein and treated as indicated. Cells were counterstained with CellMask (cytosol/plasma membrane, red). Scale bar: 25 μm. e Representative fluorescence micrographs of CASP1-positive cells (green). The fluorescent CASP1 inhibitor was added during the inflammasome induction period (16 h). Nuclei were stained with 1 μg/ml Hoechst 33342. Scale bar: 100 μm. Data in the graph are expressed as % of CASP1-positive cells (green) over total Hoechst-stained cells (blue). (n = 3 independent experiments). f Levels of IL-1β in the cell culture supernatants. Values were normalized to the total protein content of the corresponding cellular extracts. In some cases, to assess the presence of IL-1β encapsulated in EV, supernatants were incubated with 1% Triton X-100 (n = 6–7 independent experiments). g Cell death by the LDH assay. Results are expressed as % to the untreated control values (n = 6 independent experiments). Two-tailed Student's t-test (a and b) and one-way ANOVA followed by the Tukey–Kramer test (c-g) were applied to calculate statistical significance (*P ≤ 0.05, **P ≤ 0.01). See Additional file 1: Fig. S5 for uncropped blots