BMDM prepared from wild-type mice were primed with LPS (0.5 μg ml−1) for 4.5 h, then treated with or without nigericin (Ng; 20 μM) for 30 min. Necrosulfonamide (NSA) was added 3 h after LPS addition as indicated. Immunoblot analysis was used to detect released HMGB1 and cleaved IL-1β (p17) into the cell culture supernatant (a). LDH present in the extracellular media (supernatant) was quantified as a measure of pyroptosis-induced cytotoxicity. Data shown from n = 6 independent experiments (b). NSA inhibits HMGB1 and IL-1β release, as well as cytotoxicity. BMDM prepared from wild-type mice or gasdermin D knockout (Gsdmd−/−) BMDM were primed with LPS for 4.5 h, then treated with nigericin (Ng; 20 μM) for 30 min. NSA was added 3 h after LPS addition as indicated. LDH present in the extracellular media was quantified as a measure of pyroptosis-induced cytotoxicity (c). Data in c indicate n = 3 independent experiments. Immunoblot analysis of released HMGB1 and cleaved IL-1β (p17) into the cell culture supernatant, showing the loss of HMGB1 or IL-1β release in response to LPS + Ng in Gsdmd−/− BMDM (e). BMDM lysates from WT and Gsdmd−/− animals were blotted for gasdermin D with GAPDH as a loading control (d). Immunofluorescence of WT and Gsdmd−/− (f). BMDM primed with LPS and treated with or without nigericin (Ng). Green and red channels correspond to HMGB1 and red ASC oligomerized into inflammasome specks (white arrows), respectively. DAPI was used to identify cell nuclei. Note that ASC positive Gsdmd−/− cells retain nuclear HMGB1. Data with error bars are represented as mean ± SEM. Each panel is a representative experiment of at least three independent experiments. *p < 0.0001, n.s. not significant as determined by ANOVA with Tukey’s multiple comparison correction, two-sided.