BMDM were primed with LPS (0.5 μg ml−1; 4.5 h) and then treated with nigericin (Ng; 20 μM for 30 min) or potassium depletion (K+ dep; 2 h) in the absence or presence of 5 mM glycine as described in “Methods”. Untreated controls were not primed with LPS. a LDH present in the extracellular media (supernatant) was quantified as a measure of pyroptosis-induced cytotoxicity. Glycine effectively prevents cytotoxicity induced by LPS + Ng or K+ dep. Data shown from n = 3 independent experiments. b Immunoblot analysis of released HMGB1 and cleaved IL-1β (p17) into the cell culture media (supernatant) under the conditions tested in a. Glycine decreases HMGB1 release without significantly affecting IL-1β secretion. Quantitation of Western blots in b. Data in c and d shown from n = 3 independent experiments. BMDM were treated with or without LPS, then treated with PGPC (50 μg ml−1) for 4 h, or were infected with Δoat S. aureus for 18 h at the MOI indicated, as described in the Methods. Cells treated with Δoat S. aureus were not primed with LPS. e LDH release under the indicated conditions. Data in e show n = 4 (untreated, PGPC, PGPC + LPS) or n = 3 (ΔOAT10, ΔOAT30, LPS + Ng) independent experiments. IL-1β (f) and HMGB1 (g) present in the extracellular media were quantified by ELISA. Both PGPC and Δoat S. aureus stimulate IL-1β release, but not HMGB1 secretion. Data shown in f from untreated (n = 7), PGPC, LPS + PGPC, ΔOAT10, ΔOAT30 (all n = 4), LPS + Ng (n = 5) independent experiments. Data shown in g from n = 3 independent experiments. h, i Immunofluorescence of HMGB1 and DAPI, indicating nuclear retention of HMGB1 under the above conditions, and loss of HMGB1 following LPS + Ng. Data with error bars are represented as mean ± SEM. Each panel is a representative experiment of at least three independent experiments. Adjusted p values, provided in the panels, and n.s. not significant as determined by ANOVA with Tukey’s multiple comparison correction, two-sided.