BMDM were primed with LPS (0.5 μg ml−1 for 4.5 h) and then treated with nigericin (Ng; 20 μM for 30 min) or potassium depletion (K+ dep; 2 h) as described in “Methods”. Untreated controls were not primed with LPS. a Immunoblot analysis of released HMGB1, GAPDH, and cleaved IL-1β (p17) in the cell culture media (supernatant). Dashed line indicates removed lanes. Both LPS + Ng and LPS + K+ dep result in the release of HMGB1, GAPDH, and processed IL-1β into the supernatant. Data depict n = 3 independent experiments. b LDH present in the extracellular media (supernatant) was quantified as a measure of pyroptosis-induced cytotoxicity. Data are expressed as supernatant LDH as a % of total LDH from lysates and supernatants, from n = 4 independent experiments. c Immunofluorescence analysis of HMGB1 (green) and ASC (red) in BMDM treated as indicated. ASC oligomerized into inflammasome specks (white arrows) in LPS + Ng treated cells. DAPI was used to identify cell nuclei; size bar 20 μm. Data with error bars are represented as mean ± SEM. Each panel is a representative experiment of at least three independent experiments. *p < 0.0001 and n.s. not significant as determined by ANOVA with Tukey’s multiple comparison correction, two-sided.