Fig. 6

Cholesterol-enriched SH-SY5Y cells display increased susceptibility to Aβ-induced pyroptosis, which is prevented by GSHee treatment. After treatment with the CHO:MCD complex for 1 h, cells were exposed to Aβ (10 μM) for 24 h. In some cases, cells were pre-treated with GSH ethyl ester (GSHee, 4 mM) or a cell-permeable CASP1 inhibitor (10 μM) for 30 min before Aβ treatment. a Analysis of cell death by the LDH assay. LDH activity was determined in the cell culture media and normalized to total cellular LDH content. Results are expressed as % of untreated control values. (n = 6 independent experiments). b Intracellular ROS generation assessed by DCF fluorescence intensity (A.U.: arbitrary units). (n = 6–8 independent experiments). c Representative confocal immunomicrographs showing apical GSDMD puncta (white arrows) in cholesterol-enriched cells after Aβ exposure. Nuclei were stained with DRAQ5 (blue). Cells incubated with LPS (100 ng/ml) for 6 h followed by nigericin (NG, 10 μM) for 2 h were used as positive controls. Scale bar: 15 μm. Data in the graph are expressed as % of cells with GSDMD puncta over total cells. (n = 3 independent experiments). d Time-lapse microscopy of cells expressing mNeoGreen-GSDMD. Image series (20-min frames) depict the 2 h leading up to the loss of membrane integrity and cell round-up characteristic of necroptotic death in cholesterol-enriched cells exposed to Aβ. Presence of GSDMD puncta is indicated by white arrows. See Additional file 3, Additional file 4, Additional file 5 and Additional file 6 for the corresponding movies. One-way ANOVA followed by the Tukey–Kramer test was applied to calculate statistical significance (*P ≤ 0.05, **P ≤ 0.01)