GSDMD N-Terminal Antibody(Mouse specific) - #DF13758

Fig. 4. In vitro evaluation of SDT-mediated PANoptosis and immune response activation. (a) Western blotting analysis of PANoptosis-related proteins in CT26 cells after different treatments. (b) Schematic illustration of SDT-mediated PANoptosis in tumor cells. MMP, mitochondrial membrane potential. US, ultrasound. (c) Schematic illustration of the experiment of DCs maturation. (d) CLSM images of CRT expression on CT26 cells treated with different therapies. CRT: calreticulin, Scale bar: 20 μm. (e) ATP and (f) HMGB1 released from CT26 cells after different treatments. ATP, adenosine triphosphate. HMGB1, high mobility group box-1 protein. (g) Flow cytometry plot and (h) quantification of DCs incubated with CT26 cells after different treatments in transwell chambers. (i–k) ELISA results of levels of (i) IFN-γ, (j) TNF-α, (k) IL-6 cytokines in the supernatant of bone marrow-derived DCs incubated with pretreated CT26 cells. P, PBS. Na, Na2S2O8. Ca, CaS2O8. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, no significance.

Figure 6.PD‐1+CD8+ T cells from dnTGFβRII Aire−/− mice induce GSDMD mediated pyroptosis of hepatocytes. A) Detection of the expression levels of GSDMD, caspase‐1 and IL‐1β in the livers of Aire−/−, TG Aire+/− and TG Aire−/− mice by Western blot using anti‐GSDMD antibody (sc‐393581), anti‐caspase‐1 antibody (ab179515) and anti‐IL‐1β antibody (ab9722). B) Survival curves of TG Aire−/− mice treated in vivo with (n = 10) or without (n = 10) 50mg kg−1 disulfiram (DSF) by intraperitoneal injection every day from 7 days of age. C) Liver histology results of DSF treated (4‐week‐old) or untreated (about 2‐week‐old) TG Aire−/− mice. Scale bar in the upper row, 500 µm; scale bar in the bottom row, 100 µm. D) Immunohistochemistry results of the expressions of N‐GSDMD in the liver tissues from Aire−/− and TG Aire−/− mice. Scale bar, 100 µm. E) 1 × 105 PD‐1+CD8+ T cells were co‐cultured with 1 × 104 isolated primary hepatocytes and the cell morphology was observed by high content imaging system. Blue arrows reflect PD‐1+CD8+ T cells and red arrows demonstrate hepatocytes in the process of pyroptosis. Scale bar, 50 µm. F) Detection of the expression levels of GSDMD in AML12 cells with/without GSDMD knockout by Western blot using anti‐GSDMD antibody (ab219800). G) 1 × 105 CD8+ T cells isolated from the livers of dnTGFβRII Aire−/− mice were co‐cultured with 1 × 104 AML12 cells with/without GSDMD knockout and the cell morphology was imaged using a confocal microscope after 24 h. Scale bar in the left column, 100 µm; scale bar in the right column, 50 µm. H) The cell culture supernatants from (G) were detected the LDH levels and analyzed for cytotoxicity at 24 h. I) The cultured AML12 cells in (G) were labeled with FAM‐FLICA caspase‐1 and PI after 24h, then the cells were digested and detected the fluorescence signal using flow cytometry. Flow cytometry results show the fluorescence signal intensity of FLICA caspase‐1 and PI. WT co and KO co refer to wild type and GSDMD knockout AML12 cells which co‐cultured with PD‐1+CD8+ T cells respectively. J) Statistical analysis of the percentage of FLICA caspase‐1+ AML12 in total AML12 cells. Data are means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001, by log‐rank survival analysis (B) or unpaired Student's t test (H) or one‐way ANOVA (J).

Fig. 5. WFA attenuates microglial pan-PCD and promotes neuroprotection in vitro. (A) Schematic representation of BV2 microglial treatments: vehicle group (PBS treatment), LPS + adenosine group (LPS followed by adenosine treatment), and WFA group (LPS + adenosine followed by WFA intervention). (B) Quantification of apoptotic microglial cells (B2 + B4) (%) via flow cytometry. (C) Volcano plot of gene expression differences between WFA-treated and LPS + adenosine-treated groups. Specific GSEA pathways (Neurotransmitter secretion and Thanatoset) enriched in each group are displayed below. (D) GSEA enrichment comparing WFA-treated microglia to LPS + adenosine-treated microglia. (E) Heatmap of Thanatoset gene expression across vehicle, LPS + adenosine, and WFA groups. The upper panel shows Z scores (scaled FPKM values), while the lower dot plot highlights genes with log2|FC| > 1 and P < 0.05, where yellow represents up-regulated genes and blue represents down-regulated genes. (F) Western blot analysis of GSDMD-N, CASP8, Cleaved-CASP8, and CD68 protein levels across groups, demonstrating reduced protein levels in WFA-treated groups compared to LPS + adenosine groups. (G) Quantification of CTSL+ microglial area across groups (n = 3). (H) Western blot analysis of NF-κB pathway components (TLR2, p-P65, IκBα, and p-IκBα). (I) ELISA analysis of inflammatory cytokines (IL-1β, TNF-α, and IL-6) in culture supernatants (n = 5). (J) Schematic of the Transwell coculture system used to assess the effects of WFA-treated microglia on neurons. (K) Representative immunofluorescence images of neurons cocultured with microglia, showing NEUN and TUNEL costaining across groups. (L) Quantification of TUNEL+ and NeuN+ colocalization ratios (n = 3). (M) Sholl analysis of DRG neuron explants cocultured with 3 different microglial groups. Statistical analysis was performed using one-way ANOVA with Tukey (B, G, I, and L).

Fig. 4. Cur/OBs regulates macrophages polarization via PPARγ/NF-κB signaling pathway. (A, B) CLSM images showing RAW264.7 cells incubated with different drugs. Scale bar: 20 μm. (C–E) The level of TNF-α,IL-1β and IL-6 in M1 macrophages treated with different drugs was determined using ELISA. (F) Volcano plot showing the genes regulated by the treatment of Cur/OBs. (G) KEGG pathway enrichment analysis of differentially expressed genes in M1 group and M1+Cur/OBs treatment group. (H) Western blotting showing the expression of PPARγ,NF-κb p65 and Phospho-NF-κB p65 with various treatment groups. (I) Schematic diagram of the mechanism by Cur/OBs regulate the macrophages polarization via PPARγ/NF-κB signaling pathway. Data are presented as mean ± SD.

Fig. 3. RelA binds to Nlrp3 promoter and upregulates its expression in activated microglia. (A) Western blot analysis of NLRP3, RelA, Caspase1 and GSDMD-N protein expression levels in BV2 cells treated with PBS (control) or LPS (1 μg/mL) + ATP (3 mM) for 4 h and the corresponding quantification. The inter-group comparison was performed using an independent samples t-test. Statistical difference was defined as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Data represent mean ± SEM from three independent biological replicates. (B) Computationally predicted sequence of RelA binding sites within Nlrp3 promoter region using JASPAR (http://jaspar.genereg.net). (C) Luciferase reporter assay in BV2 microglia transfected with Nlrp3 WT luciferase constructs. The inter-group comparison was performed using an independent samples t-test. Statistical difference was defined as ***p < 0.001. Data represent mean ± SEM from three independent biological replicates. (D) Relative Nlrp3 promoter enrichment measured by ChIP-qPCR. Enrichment was calculated relative to IgG and presented as fold change. The inter-group comparison was performed using an independent samples t-test. Statistical difference was defined as ***p < 0.001. Data represent mean ± SEM from three independent biological replicates. (E) Luciferase reporter assay assessing Nlrp3 promoter activity in BV2 microglia. (F) Luciferase reporter assay assessing Nlrp3 promoter activity in primary microglia. Microglia were transfected with Nlrp3 WT, MutA, or MutB luciferase reporter constructs, along with the pTK-Renilla luciferase plasmid as an internal control. Luciferase activity was measured 48 h post-transfection and normalized to Renilla luciferase activity. The inter-group comparison was performed using an independent samples t-test. Statistical difference was defined as ns, not significant, *p < 0.05, and **p < 0.01. Data represent mean ± SEM from three independent biological replicates.

Fig. 3. CLBRP affects intracellular Ca2+ levels and induces pyroptosis. (A) Schematic illustration of CLBRP-mediated pyroptosis (created in BioRender.com). (B, C) Fluorescence images and intracellular Ca2+ levels detected by the Fluo-4 AM probe were quantified in cells (scale bar = 25 μm). (D) Differences in Na+/K+-ATPase activity after treatment with different drugs. (E, F) Mitochondrial membrane potential measured by JC-1 and corresponding quantitative analysis in cells (scale bar = 50 μm). IL-18 (G) and IL-1β (H) release in the different groups. (I, J) Caspase-1 immunofluorescence images and corresponding quantitative analysis of cells following various treatments (scale bar = 50 μm). (K) Western blot of the pyroptosis-related proteins caspase-1, cleaved caspase-1, and GSDMD-N (n = 6). The data are shown as the means ± SDs. Statistical significance was determined by one-way ANOVA with Tukey's two-tailed LSD multiple-comparisons test: ns, not significant; ∗P < 0.05, ∗∗P < 0.01.