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).

Impacts of circPRKAR1B and m6A modification on NLRP3 inflammasome‐mediated pyroptosis. The assessment of the impacts of si‐circPRKAR1B and si‐METTL3 treatment on NLRP3 inflammasome‐mediated pyroptosis using western blotting (A)