Figure 4.

Reduction of tumor necrosis factor (TNF)–induced GSDME‐mediated macrophage pyroptosis by the caspase 3–specific inhibitor Z‐DEVD‐FMK. After treatment with Z‐DEVD‐FMK for 1 hour, THP‐1 cell–derived macrophages were exposed to 50 or 100 ng/ml TNF. A, Representative immunofluorescence microscopy images showing the expression of GSDME in macrophages treated as indicated. Bars = 50 μm. B, Western blot of GSMDE‐FL, GSDME‐N, and activated caspase 3 expression (left), quantification of GSDME‐N expression (middle), and quantification of activated caspase 3 expression (right) in lysates of macrophages treated as indicated. Values are the protein level relative to β‐actin C, Left, Representative fluorescence microscopy and brightfield images of macrophages treated as indicated and stained with propidium iodide (PI; red). Nuclei were counterstained with Hoechst (blue). For the merged images, the right panels show higher‐magnification views (original magnification × 1600) of the boxed areas in the left panels (original magnification × 400). Right, Percentage of PI‐positive cells. D, Left, Flow cytometric analysis of cells treated as indicated and stained with annexin V/PI. Data are representative of 3 independent experiments. Right, Percentage of PI‐positive cells. In B, C, and D, bars show the mean ± SD. * = P < 0.05; *** = P < 0.001. See Figure 1 for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.41963/abstract.