Figure 3.

Tumor necrosis factor (TNF) induction of pyroptosis of macrophages through activation of the caspase 3/GSDME pathway in vitro. THP‐1 cell–derived macrophages were seeded in 24‐well or 6‐well plates and incubated with different concentrations of TNF (ng/ml) for 36 hours or TNF and cycloheximide (CHX) (10 μg/ml; positive control) for 24 hours. A, Western blot (left) and quantification (right) of GSDME‐FL, GSDME‐N, and activated caspase 3 expression in macrophage lysates treated as indicated. Values are the protein level relative to β‐actin. B, Left, Phase‐contrast and fluorescence microscopy images of macrophages treated as indicated and stained with Hoechst (blue) and propidium iodide (PI; red). For the merged images, 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. C, Lactate dehydrogenase (LDH) release from macrophages treated as indicated. D, Flow cytometric analysis of cells stained with annexin V/PI to determine cell death (left) and percentage of PI‐positive cells (right) among macrophages treated as indicated. Data are representative of 3 independent experiments. Bars show the mean ± SD. * = P < 0.05; ** = P < 0.01; *** = P < 0.001. NS = not significant (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.