Fig 1: GSDMD-mediated pyroptosis of THP-1 cells induced by serum from SLE patients was suppressed by DSF.A Effect of DSF on the cell viability of THP-1 cells. Cells were treated with DSF (0, 1, 5, 10, 20 µM) for 48 h and then cell viability measured by CCK-8 assay. B The morphological features of THP-1 cells treated with serums from healthy controls or SLE patients, with or without mixing 10 µM disulfiram. C Lactate dehydrogenase (LDH) release from THP-1 cells treated as indicated. D Hoechst33342/Propidium Iodide (PI) double staining in THP-1 cells after different treatments. E Representative immunofluorescence images showing the expression of NLRP3, caspase-1, and cleaved caspase-1 in THP-1 cells treated as indicated. F The expression of full length and cleaved GSDMD in THP-1 cells. The cells were incubated in different mediums and analyzed by western blot analysis. ß-actin was used as a protein loading control. G, H The expression level of total GSDMD and cleaved-GSDMD relative to ß-actin were quantified. Total GSDMD = full length-GSDMD + cleaved-GSDMD. Significant differences were calculated using one-way ANOVA. Values were shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was repeated three times. FBS fetal bovine serum, HC Healthy control, NS not significant, PI propidium iodide, LDH Lactate dehydrogenase.
Fig 2: Lactate Increases Pyroptosis in Human NP Cells. (A) The lactate-induced increase in IL-1ß release by human NP cells was detected by ELISA; this process was blocked by the pyroptosis inhibitors glycine and YVAD. (B) The increased LDH release by human NP cells was detected by ELISA; this process was also blocked by glycine and YVAD. (C-E) Protein levels of GSDMD-N and IL-1ß in human NP cells were examined by immunoblotting. (F, G) Pyroptosis level of human NP cells was examined by flow cytometry. (H, I) Immunofluorescence staining confirmed the death level of lactate-stimulated human NP cells. Scale bar = 50 µm. Data are represented as mean ± SD (n = 3). Significant differences between groups are indicated as *P < .01, compared with control group; **P < .01, compared with lactate group
Fig 3: Tanshinone IIA enhances pyroptosis of HK1 cells. (A) Reverse transcription-quantitative PCR analyses of the relative mRNA levels of caspase-1 and GSDMD in HK1 cells. Gene expression was normalized to the ß-actin mRNA level. (B and C) Representative western blot analysis and quantification of cleaved caspase-1 and GSDMD in HK1 cells. Protein expression was normalized to ß-actin levels. (D) Relative ROS levels in HK1 cells after tanshinone IIA treatment. (E) Relative levels of LDH release in HK1 cells after tanshinone IIA treatment. (F) Enzyme-linked immunosorbent assay of IL-1ß and IL-18 in HK1 cells after tanshinone IIA treatment. *P<0.05; **P<0.01; ***P<0.001. All experiments were repeated at least three times. GSDMD, gasdermin D; ROS, reactive oxygen species; LDH, lactate dehydrogenase; IL, interleukin.
Fig 4: The NF-?B inhibitor PDTC rescued the function of HMBOX1 in AC16 cells. AC16 cells with HMBOX1 silencing (siHMBOX1) or not (siNC) were treated with NF-?B inhibitor PDTC (10 µM) for 24 h. (A) PI+caspase-1+ pyroptosis was measured by flow cytometry. Representative images were shown on the left and summarized results are on the right. (B) Supernatant levels of IL-1ß and IL-18 were measured by ELISA. The protein levels of HMBOX1, NF-?B (cytoplasm), NF-?B (nuclear) (C) and NLRP3, caspase-1, GSDMD-N (D) were measured by western blot. (A,B) Data are mean ± SD from three independent experiments (n = 3 per group). * p < 0.05.
Fig 5: Upregulation of TUG1, TET2, and Inflammation and Downregulation of miR-200a-3p Under OGD/R Conditions. A: Cell viability was determined by MTT assay after deoxygenation for 0, 2, 4, 6, 8, 12 h, then following by reoxygenation for 24 h. B: TUG1, miR-200a-3p, NLRP3, and TET2 were detected by qRT-PCR after subject to ORG 12 h/R 24 h condition. C: IL-1ß and IL-18 were measured by ELISA after subject to ORG 12 h/R 24 h condition. D: TET2, NLRP3, Caspase-1, GSDMD-N, IL-18, and IL-1ß were determined by western blot after subject to ORG 12 h/R 24 h condition. Four independent experiments were repeated, with three replicates each time. *p < 0.05, **p < 0.01, ***p < 0.001.
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