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. β-actin was used as a protein loading control. G, H The expression level of total GSDMD and cleaved-GSDMD relative to β-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‐1β 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‐1β 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 β-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 β-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-1β 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-κB inhibitor PDTC rescued the function of HMBOX1 in AC16 cells. AC16 cells with HMBOX1 silencing (siHMBOX1) or not (siNC) were treated with NF-κB 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-1β and IL-18 were measured by ELISA. The protein levels of HMBOX1, NF-κB (cytoplasm), NF-κB (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-1β 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-1β 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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