Fig. 4

Cholesterol-enriched neuroblastoma SH-SY5Y cells show enhanced endotoxin-mediated activation of NLRP3 inflammasome. After treatment with the CHO:MCD complex cells were co-incubated with LPS and MDP (L + M, 10 μg/ml each for 16 h) or MDP (10 μg/ml, 16 h) plus ATP (5 mM, 1 h). a Western blot analysis of NLRP1 and NLRP3 in cellular extracts. (n = 3 independent experiments). b Representative confocal images of oligomeric ASC. Cells were transfected with a plasmid encoding an ASC:GFP fusion protein and 48 h later were treated as indicated. Cells were counterstained with CellMask (cytosol/plasma membrane, red) and DRAQ5 (nuclei, blue). Scale bar: 25 μm. Data in the graph are expressed as % of speck-positive cells of total transfected cells (n = 3 independent experiments). c Representative immunoblots from 3 independent experiments showing pro- and cleaved CASP1 (active product of 20 kDa) in cellular extracts. d Representative micrographs of CASP1-positive cells (green). The fluorescent CASP1 inhibitor was added during the inflammasome induction period (16 h). Nuclei were stained with Hoechst 33342 (1 μg/ml). Scale bar: 100 μm. Data in the graph are expressed as % of CASP1-positive cells to the total Hoechst-stained cells (blue) (n = 3 independent experiments). e Representative immunoblots of pro- and mature IL-1β in cellular extracts. (n = 4 independent experiments). f Levels of IL-1β in the cell culture supernatants. Values were normalized to the total protein content and expressed as % relative to the untreated control values (n = 4 independent experiments). g Cell death by the LDH assay. Results are expressed as % to the untreated control values (n = 4 independent experiments). In western blots, ACTB/actin β and ponceau S (PS) staining were used as loading controls and optical density (O.D.) values of the bands representing the specific protein immunoreactivity were normalized to PS staining. One-way ANOVA followed by the Tukey–Kramer test was applied to calculate statistical significance (*P ≤ 0.05, **P ≤ 0.01). See Additional file 1: Fig. S6 for uncropped blots