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. 2015 Nov 27;25(12):1285–1298. doi: 10.1038/cr.2015.139

Figure 4.

Figure 4

GSDMD can be cleaved by pro-caspase-1. (A) GSDMD is cleaved in ASC-deficient cells in the absence of pro-caspase-1 processing. Gsdmd−/− RAW264.7 and Gsdmd−/− RAW-asc cells reconstituted with the expression of Flag-GSDMD, GSDMD-Flag or a control vector were primed with LPS for 4 h followed by S. typhimurium (100 MOI) treatment for 2 h. The cells were analyzed by immunoblotting with anti-caspase-1 and anti-Flag antibodies. (B) LDH release was measured in the cells described in A. (C) The auto-cleave-deficient pro-caspase-1 mutant can cleave GSDMD. Gsdmd and caspase-1 double knockout RAW-asc cells (Gsdmd−/− Casp1−/−) were reconstituted with GSDMD-Flag expression first and then with WT, auto-cleavage-deficient mutant D6N, and enzymatic dead mutant C284A of pro-caspase-1, respectively. The cells were treated as in Figure 1B and analyzed by immunoblotting with anti-caspase-1 and anti-Flag antibodies. (D) Pyroptosis can be mediated by auto-cleavage-deficient pro-caspase-1. The cells described in C were stained with PI and analyzed under microscope. (E) IL-1β production cannot be mediated by auto-cleavage-deficient pro-caspase-1. IL-1β in the culture media of the cells described in C was measured by ELISA. Graphs show mean ± SD of triplicate wells and represent three independent experiments.