Figure 1
From: Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion
Identification of GSDMD as a component of NLRP3 inflammasome and determination of the requirement of GSDMD in LPS plus nigericin-induced pyroptosis and IL-1β production. (A) Finding protein candidates that were recruited to NLRP3 complex by quantitative MS. NLRP3 complex was affinity-purified from NLRP3-Flag J774 cells after LPS (100 ng/ml) priming for 4 h and then nigericin (10 μM) stimulation for 0, 5, 15, 40, 60, or 90 min, digested with trypsin and subjected to high-sensitive quantitative MS analysis. MS data was analyzed with Group-DIA and peptide intensities were extracted. The results revealed nine candidate proteins whose amount time-dependently increased in NLRP3 complex. The extracted ion chromatogram (XIC) peaks of multiple product ions of one representative peptide for each of these proteins were shown. Arrows indicate the co-eluting XIC peaks of product ions of corresponding representative peptides. (B) GSDMD is involved in inflammasome activation. The genes as indicated were knocked out by CRISPR-Cas9 in RAW-asc cells. The cells were primed with LPS for 4 h and then treated with nigericin for 2 h. The release of p20 caspase-1 into the culture media was measured by immunoblotting using anti-caspase-1 antibody, which was used to assay pyroptosis. (C) Time-dependent recruitment of GSDMD and caspase-1 to NLRP3 complex. Relative abundance of NLRP3, caspase-1 and GSDMD proteins in NLRP3 immunocomplex across six time points was shown. (D) Requirement of GSDMD in pyroptosis. LDH released from RAW-asc cells with different gene deletion or gene reconstitution was measured after the cells were treated as in B. RAW-asc cells with genotype of WT, Nlrp3−/−, Caspase-1−/−(Casp1−/−), Gsdmd−/− cells reconstituted with N-terminally Flag-tagged GSDMD (Flag-GSDMD) or C-terminally Flag-tagged GSDMD (GSDMD-Flag) or a control vector were used in the experiments. (E) Requirement of GSDMD in IL-1β production. Culture supernatants of the cells described in D were analyzed by IL-1β ELISA kit. (F) Pyroptosis and IL-1β in the culture supernatants of WT and Gsdmd−/− BMDM were measured as in D, E. (G) Pyroptosis and IL-1β in the culture supernatants of WT and Gsdmd−/− J774 cells were measured as in D and E. Graphs show mean ± SD of triplicate wells and represent three independent experiments.
