Figure 3
From: Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion
Cleavage of GSDMD at D276 is required for pyroptosis and IL-1β secretion and the N-terminal proteolytic fragment of GSDMD executes cell death. (A) Processing of GSDMD. WT RAW-asc cells and Gsdmd−/− RAW-asc cells reconstituted with Flag-GSDMD, GSDMD-Flag or a control vector were treated as in Figure 1B. Cell extracts together with their corresponding culture supernatants or supernatants alone were analyzed by immunoblotting with anti-Flag antibody. (B) D276 is the cutting site in GSDMD upon NLRP3 inflammasome activation. Gsdmd−/− RAW-asc cells reconstituted with the expression of GSDMD-Flag, D276N mutant of GSDMD (D276N-Flag) or D276A mutant of GSDMD (D276A-Flag) were treated as in Figure 1B. Cell extracts together with their corresponding culture supernatants were analyzed by immunoblotting with anti-Flag antibody. (C) Requirement of GSDMD cleavage in pyroptosis. LDH release was measured in the cells described in B. (D) Requirement of GSDMD cleavage in IL-1β secretion. The same as in C except that IL-1β in the culture media was measured by ELISA. (E) Pyroptosis induced by N-terminal domain of GSDMD. Green fluorescence protein (GFP) and one of the GSDMD fragments were co-expressed in 293T cells and images were taken 16 h after transfection. Graphs show mean ± SD of triplicate wells and represent three independent experiments.
