Fig. 6

Activated caspase-3/7 accumulate in the nucleus and promote nuclear shrinkage during pyroptosis. (a) Human microglia were exposed to nigericin (5.0 μM, 4 h), fixed, and immunolabelled for GSDMD (green) and either cleaved caspase-3 or -7 (amber). Images shown are representative 3-dimensional z-stacks incorporating 15 XY planes over a vertical distance of 4–6 μm. One square unit represents 10.28 μm. (b–e) Mean fluorescence intensity (MFI) of cleaved caspase-3 (b, c) and caspase-7 (d, e) in the nucleus was assessed in unexposed (n = 50) and nigericin-exposed (minimum n = 150) microglia and categorized by pyroptotic stages. Data shown represent mean nuclear cleaved caspase-3/7 MFI ± SEM for a representative human donor. Results were independently verified in microglia from 2 to 3 separate human donors (**p < 0.001, Student’s t test). (F) Microglia were exposed to staurosporine (5 μM, 4 h), nigericin (5 μM, 4 h) or ATP (100 μM, 24 h), fixed and DAPI-stained. Cross-sectional area (μm²) of DAPI-stained nuclei were measured (n = 10–20 cells per condition). Data shown represent mean nuclear area ± SEM for a representative human donor. Data were tested for significance using one-way ANOVA with Dunnett’s test for multiple comparisons (****p < 0.0001). (G) Microglia were transfected with either universal non-coding siRNA (NC siRNA) or a cocktail of different siRNAs targeting caspases-3 and -7 (Casp-3/7 siRNA) prior to nigericin exposure (5 μM, 24 h). Cells were fixed, DAPI stained to label nuclei, and assessed by confocal microscopy. Cross-sectional area (μm²) of DAPI-stained nuclei were measured in unexposed (n = 20) and nigericin-exposed (minimum n = 50) microglia. Data shown represent mean nuclear area ± SEM for a representative human donor (***p < 0.001, ****p < 0.0001, analyzed using one-way ANOVA with Dunnett’s test for multiple comparisons). (H, I) Cells were transfected with either universal non-targeting siRNA (NT siRNA) or a cocktail of different siRNAs targeting caspase-3 and -7 (Casp-3/7 siRNA) prior to (H) nigericin (5 μM, 24 h) or (I) ATP (100 μM, 24 h) exposure. Total DAPI levels within each population were assessed fluorometrically by microplate reader, with a minimum of n = 8 technical replicates per condition (**p < 0.01, ***p < 0.001, ****p < 0.0001, analyzed using one-way ANOVA with Dunnett’s test for multiple comparisons).