Fig 1: H3 relaxin inhibits CaOx crystal‐induced pyroptosis by preventing the reactive oxygen species (ROS)‐mediated NLRP3 inflammasome activation. A, Western blot analysis of related proteins: NLRP3, ASC, IL‐18 and IL‐1β in the treated tubular epithelial cells (TECs) or rat kidney samples (14 d), in the absence or presence of H3 relaxin. B, The protein expression of NLRP3 and cleaved GSDMD was detected using immunofluorescence assays in the treated TECs in groups. Scale bars: 100 μm. C, The protein expression of NLRP3 was detected using immunohistochemical assays in the treated rat kidneys (14 d), with or without H3 relaxin treated. Scale bars: 50 μm. D, Western blot analysis of inflammation‐related proteins: NLRP3, ASC, cleaved caspase‐1, cleaved GSDMD, IL‐18 and IL‐1β in the treated TECs, in the absence or presence of H3 relaxin and Mcc950. n = 3 per group, representative images are shown. E, ROS generation of the treated TECs was detected by DCFH‐DA probe using flow cytometric analysis. n = 3, data expressed as means ± SEM, **P < .01. F, Western blot analysis of inflammatory pyroptosis‐related proteins in the treated TECs, in the absence or presence of H3 relaxin and H2O2. G, Western blot analysis of inflammatory pyroptosis‐related proteins in the treated TECs, in the absence or presence of H3 relaxin and NAC. H, Western blot analysis of inflammatory pyroptosis‐related proteins in the treated TECs, in the absence or presence of H2O2 and MitoTEMPO. n = 3 per group, representative images are shown
Fig 2: Peritoneal macrophages have distinct morphological features of pyroptosis after SEZ infection. (A) Representative propidium iodide (PI) staining of GSDMD+/+ or GSDMD-/- macrophages after being infected or uninfected with SEZ. (B) Representative scanning electronic microscopy (SEM) images of GSDMD+/+ or GSDMD-/- macrophage cells are treated as above, and the arrow points to the bubbling of pyroptotic cells. Scale bar, 70 μm (A). These experiments were conducted with 3 replicates.
Fig 3: GSDMD deficiency results in enhanced pathology but not macrophages number in the spleen during SEZ infection. GSDMD+/+ and GSDMD-/- mice (n=3) were treated with SEZ (106 CFU) or equivalent PBS for 24 h. The black arrow marks the multinucleated macrophages. The yellow arrow indicates hemosiderin. The white arrow points to the clear white pulp of the spleen. One representative experiment of three is shown. Scale bar, 25 μm (A, B) The number of multinucleated macrophages was calculated in the microscopic fields of the slides. (C) The proportion of hemosiderin area to total area was calculated by randomly selecting three microscopic fields. These experiments were conducted with 3 replicates. Data are expressed as mean ± SD. ns, not significant, **p<0.01, one-way ANOVA (B, C).
Fig 4: Metformin can decrease chondrocyte pyroptosis in a destabilization of the medial meniscus model at the protein level. (A) NLRP3, (B) caspase-1, (C) GSDMD and (D) IL-1β immunohistochemistry test results (magnification, x400). Scale bar=100 µm. Black arrows indicate the positive cells. The ratio of positive cells immunoreactive for (E) NLRP3, (F) caspase-1, (G) GSDMD and (H) IL-1β. One-way ANOVA followed by Tukey's multiple comparison test was used to compare data among groups after testing the data for homogeneity of variance. ***P<0.001; ###P<0.001. NLRP3, NOD-like receptor protein 3; GSDMD, gasdermin D.
Fig 5: Hypoxia-preconditioned OM-MSCs downregulated the level of molecules closely related to pyroptosis. (A) Western blotting results of NLRP3, ASC, cleaved caspase-1, cleaved caspase-8, GSDMD, and mature IL-1β in the cortex of the perihematomal regions at 3 days post-ICH. (B) Quantitative analysis of target proteins. Data are expressed as the mean ± SEM (n=3) (**, P<0.01; ***, P<0.001). Abbreviations: OM-MSCs, olfactory mucosa mesenchymal stem cells; ICH, intracerebral hemorrhage; NLRP3, nod-like receptor family protein 3; ASC, apoptosis-associated speck-like proteins containing a caspase recruitment domain; GSDMD, Gasdermin D; SEM, standard error of mean.
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