Fig. 2.

DSS augments ATP- or nigericin-induced NLRP3 inflammasome activation and pyroptosis in macrophages. Bone marrow-derived macrophages (BMDMs) were differentiated from the bone marrow cells of C57BL/6J mice. After being primed with LPS (0.5 μg/mL) for 4 h, the cells were treated with DSS for 1 h followed by ATP (2 mM, 30 min) and nigericin (1 µM, 1 h) stimulation. The indicated proteins in precipitated proteins from culture supernatants (SN) and cell lysates were examined by Western blot analysis. β-Actin was used as a loading control for cell lysates. A, B DSS dose-dependently enhanced inflammasome activation triggered by ATP (A) or nigericin (B). The arrowhead denotes nonspecific bands. C, D DSS promoted the release of IL-1β from macrophages stimulated with ATP (C) or nigericin (D). The levels of IL-1β in the supernatants were measured with a cytometric bead assay. E DSS changed the morphological characteristics of dying cells. Cell death was determined by PI/Hoechst 33342 staining. One set of representative images from three independent experiments is shown. Scale bars, 50 µm. F, G DSS dose-dependently increased the percentage of lytic cell death triggered by ATP (F) or nigericin (G). The ratios of PI-positive cells in 5 randomly chosen fields were quantified. The data are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001