Fig. 3.

NLRP3 inhibition and depletion abrogate the DSS-induced increase in ATP-induced inflammasome activation. BMDMs derived from Nlrp3^−/− mice and their Nlrp3^+/+ littermates were differentiated and treated as described in Fig. 1. Cells were pretreated with a specific inhibitor and DSS and then stimulated with ATP for 30 min. Western blotting was used to examine the indicated proteins in cell lysates and precipitated proteins from culture supernatants (SN). IL-1β levels in culture supernatants were measured by CBA. Lytic cell death was determined by PI/Hoechst 33342 staining, and the ratios of PI-positive cells in 5 randomly chosen fields were quantified. In addition, merged images showing PI and Hoechst 33342 fluorescence and bright-field images are shown. Scale bars, 50 µm. A–C MCC950 or VX-765 treatment blocked DSS-mediated enhancement of ATP-induced inflammasome activation (A), IL-1β secretion (B) and lytic cell death (C) in WT cells. D–F NLRP3 depletion blocked ATP-induced inflammasome activation (D), IL-1β secretion (E) and lytic cell death (F) in the presence of DSS. The data are expressed as the mean ± SD. ***p < 0.001