Figure 6. Outcomes of inflammasome activation in astrocytes.

(A and B) RT-qPCR evaluation of gene expression for Il1b and Il18 (A) and Casp1 and Gsdmd (B) in SC total cells versus astrocytes isolated from naive mice (n = 4) and mice with EAE at 30 dpi (n = 7). Two-way RM ANOVAs were used with Holm-Šidák multiple comparisons test post hoc. Each data point represents a value from 1 mouse. (C) Representative images of Western blotting for inflammasome components in BMDMs versus primary cortical astrocytes. Cells indicated as group 1: unstimulated; group 2: treated with ultrapure LPS alone; group 3: pretreated with ultrapure LPS and stimulated with nigericin to activate the NLRP3 inflammasome; and group 4: pretreated with ultrapure LPS and stimulated with poly(dA:dT)/liposome to activate the AIM2 inflammasome. GSDMD-FL, uncleaved GSDMD; GSDMD-NT, cleaved N-terminal GSDMD. (D–F) Representative images (D and E) and quantification (F) of TUNEL staining of SC sections from naive (n = 3) and 30 dpi EAE ASC-Citrine mice (n = 4). Arrows indicate TUNEL⁺ cells (E). Two-way RM ANOVA was used (F). Scale bar is 75 μm. (G and H) Representative images (G) and quantification (H) of active caspase-3 (CC3) in SC astrocytes with and without ASC specks/strings. Evaluated from naive (n = 7) and at 30 dpi EAE (n = 6) ASC-Citrine mice. Scale bar is 15 μm. Individual astrocytes were identified using the Imaris software and were quantified by CC3 puncta staining. Each data point represents a value from 1 mouse, combined from multiple experiments. Two-way RM ANOVA was used with Holm-Šidák multiple-comparison test post hoc. (H) **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars denote mean ± SEM (A, B, F, and H).