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

FIGURE 6 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), grayscale analysis of blots (B), immunofluorescence (C; scale bar: 20 μm, white arrows: NLRP3 inflammasome specks and D; scale bar: 25 μm), TUNEL staining (E; scale bar: 25 μm), pyroptosis analysis via SEM (F; scale bar: 100 and 20 μm) and proinflammatory cytokine (IL‐1β/18) level analyses via ELISA (G). METTL3, methyltransferase‐like 3; m6A, N6‐methyladenosine; SEM, scanning electron microscopy; NLRP3, nucleotide‐binding oligomerization domain, leucine‐rich repeat and pyrin domain‐containing 3; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; ELISA, enzyme‐linked immunosorbent assay; IL, interleukin. The data are presented as mean ± SD. ***p < .001 by one‐way ANOVA.

Figure 2. Upregulation of miR-140-5p alleviated chondrocyte pyroptosis in OA mice. the OA mice were injected with agomiR-140-5p, with agomiR-NC as the control. A: cleaved caspase-1 positive expression was detected using immunohistochemical staining; the arrow indicates positive staining. B: GSDMD-N protein level was detected using western blot. C: IL-1β and IL-18 levels were detected using ELISA kits. N = 6. the measurement data are expressed as mean ± standard deviation and analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test, ***p < 0.001

Figure 3. NLRP3 knockdown ameliorates oxalate-caused injury of HK-2 cells. (A) Western blot detection of NLRP3, caspase-1, GSDMD and GSDMD-N protein contents in HK-2 cells treated with 0.8 mM oxalate for 24 h after transfection with NLRP3-siRNA. (B) Relative lactate dehydrogenase levels in HK-2 cells transfected with NLRP3-siRNA upon 0.8 mM oxalate treatment for 24 h (n=6). (C) Images of two colors (blue and green) and merged colors in HK-2 cells with TUNEL staining were observed by confocal microscopy. HK-2 cells with NLRP3 silence were treated with 0.8 mM oxalate for 24 h followed by staining. Scale bars, 100 µm. (D) Images of three colors (blue, green and red) and merged colors in HK-2 cells with Calcein-AM/PI staining were recorded through confocal microscopy. HK-2 cells transfected with NLRP3-siRNA or scrambled-siRNA were dealt with 0.8 mM oxalate for 24 h. Scale bars, 100 µm. NLRP3, leucine-rich repeat-containing family pyrin domain-containing 3; GSDMD, gasdermin D; siRNA, small interfering RNA; Ctrl, control; NC, negative control.

Figure 7. GSDMD mediates oxalate crystal-related pyroptosis in vivo. (A) Von Kossa staining revealed oxalate crystals in kidney of mice treated with Gly. (B) Haematoxylin and eosin staining demonstrated renal epithelial cell swelling, renal tubule edema and neutrophil infiltration in Gly treated mice. Scale bars, 50 µm. (C) Serum creatinine level of Gly-treated mice was examined by creatinine assay (n=6). (D) Western blot analysis of indicated proteins in NLRP3-GSDMD pathway in oxalated crystal mice. (E) The expression of same proteins was detected through immunohistochemical assays in mice. Scale bars, 50 µm. GSDMD, gasdermin D; Gly, glyoxylic acid; NLRP3, leucine-rich repeat-containing family pyrin domain-containing 3; OPN, osteopontin; Ctrl, control.