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Fig. 5 | Translational Neurodegeneration

Fig. 5

From: Inflammasome activation under high cholesterol load triggers a protective microglial phenotype while promoting neuronal pyroptosis

Fig. 5

Cholesterol overload promotes inflammasome activation in SH-SY5Y cells in response to Aβ exposure. For cholesterol enrichment, cells were treated with the CHO:MCD complex for 1 h. After 4 h of recovery, cells were exposed to Aβ (10 μM) for 24 h. In some cases, cells were pre-treated with GSH ethyl ester (GSHee, 4 mM) for 30 min before Aβ treatment. a Western blot analysis of NLRP1, NLRP3 and pro- and cleaved CASP1 (self-cleavage and active product of 20 kDa) in cellular extracts. The ACTB/actin β immunoblot 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 the values of the corresponding PS staining (n = 4 independent experiments). b Representative confocal images of oligomeric ASC forms. To monitor ASC-dependent inflammasome assembly cells were transfected with a plasmid encoding an ASC:GFP fusion protein and counterstained with CellMask (cytosol/plasma membrane, red) and DRAQ5 (nuclei, blue). Speck formation (seen as an aggregate) was determined by confocal microscopy and the number of speck-positive cells of total transfected cells was quantified (n = 3 independent experiments). Scale bar, 25 μm. c Levels of IL-1β in the cell culture supernatants after Aβ incubation (n = 4 independent experiments). One-way ANOVA followed by the Tukey–Kramer test was applied to calculate statistical significance (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). See Additional file 1: Fig. S6 for uncropped blots

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