Fig 2: NLRP3 was needed in IL6 induced pyroptosis(A) SEM morphology of FE25 cells treated with 100 µM IL-6 for 0, 24, and 36 h.(B) WB assays of NLRP3, cleaved caspase-1, and GSDMD in FE25 were treated with 100 µM IL-6 for 72 h.(C) The expression and intracellular localization of NLRP3 in FE25 cells treated with 100 µM IL-6 with or without MCC950 (NLRP3 inhibitor) and siNLRP3 were detected using confocal microscopy.(D) FE25 cells were stained with Annexin V/propidium iodide (PI) for flow cytometry after treated with 100 µM IL-6 with or without siNLRP3 for 72 h. The expression and intracellular localization of Caspase1 (E), GSDMD (F) and GSDMD-N (G) in FE25 cells treated with 100 µM IL-6 with or without MCC950 (NLRP3 inhibitor) and siNLRP3 were detected using confocal microscopy. Vehicle treated with conditioned medium of NFs treated with 1‰ DMSO.

Fig 3: IFN-? promotes HCE-T cell pyroptosis under hypertonic stress. (A) The mRNA levels of NLRP3, AIM2, ASC, CASP1, and GSDMD in control and IFN-? plus hyperosmolarity-treated HCE-T cells (n = 3/group). (B) LDH assay showing increased release of LDH in HCE-T cells treated with IFN-? plus hyperosmolarity (n = 5/group). (C) Decreased viability in HCE-T cells treated with IFN-? plus hyperosmolarity, as measured by the CCK-8 kit (n = 5/group). (D) Representative immunoblots showing NLRP3, ASC, C-CASP1, and N-GSDMD. Relative protein expression levels were normalized to GAPDH (n = 3/group). (E) Representative immunofluorescence staining of NLRP3, ASC, C-CASP1, and N-GSDMD in control and IFN-? plus hyperosmolarity-treated HCE-T cells (n = 3/group). Scale bar: 100 µm. Error bars: SD. *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001 versus untreated controls.

Fig 4: Inhibiting STAT1 suppresses pyroptosis in vitro. (A) The mRNA expression of JAK2 and STAT1 in control and IFN-? plus hyperosmolarity-treated HCE-T cells (n = 3/group). (B) Western blot and quantitative analysis of proteins related to pyroptosis in HCE-T cells (n = 3/group). (C) Representative immunofluorescence staining of JAK2, p-JAK2, STAT1 and p-STAT1 in HCE-T cells (n = 3/group). (D) The mRNA expression of pyroptosis-related genes in si-NC and si-STAT1 HCE-T cells with or without IFN-? plus hyperosmolarity treatment (n = 3/group). (E) Western blot and quantitative analysis of STAT1, p-STAT1, NLRP3, ASC, C-CASP1, and N-GSDMD in HCE-T cells. *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001 versus controls. #P < 0.05; ###P < 0.001; ####P < 0.0001 versus Hy + IFN-? + NC group.

Fig 5: Drp1 hyperactivation in mature OLs of AD models.(A) Total protein lysates were harvested from the frozen postmortem cortex of AD patients and control subjects (Table 2). Western blot analysis was performed in the absence of 2-mercaptoethanol (ß-ME) to examine Drp1 oligomers. Histogram: the relative density of Drp1 tetramer versus Drp1 monomer is shown. Student’s t test. (B) Postmortem cortex sections from normal subjects and AD patients (Table 1) were stained with anti–p-Drp1 Ser616 and anti-CC1 antibodies. Scale bar, 50 µm. The number of p-Drp1 Ser616+ CC1+ cells per square millimeter was quantified and is presented in the histogram. Student’s t test. (C) Total protein lysates from the corpus callosum of WT and 5XFAD mouse brains were harvested at the ages of 3 and 6 months. Drp1 tetramer was analyzed by Western blot analysis in the absence of ß-ME. The relative density of Drp1 tetramer versus the Drp1 monomer is shown in the histogram. n = 3 mice per group. Student’s t test. (D) Mature OLs were differentiated from OPCs and exposed to oligomeric Aß1-42 for the indicated periods. Drp1 tetramer was analyzed by Western blot analysis of total protein lysates in the absence of ß-ME. The relative density of Drp1 tetramer versus the Drp1 monomer is shown in the histogram. The data from five independent experiments were analyzed using one-way ANOVA with Tukey’s post hoc test. (E) Mature OLs were treated with oligomeric Aß1-42 for 24 hours and stained with anti–p-Drp1 Ser616 (green) and anti-MBP (red) antibodies. The intensity of p-Drp1Ser616 in the MBP+OLs was quantitated and is presented in the histogram. The data from three independent experiments were compared using the Student’s t test. Scale bar, 20 µm. (F) A linear correlation between the relative Drp1 tetramer density and the cleavage of GSDMD is shown. All data are expressed as the mean ± SEM.