BMDM were primed with LPS (0.5 μg ml−1; 4.5 h) and then treated with nigericin (Ng; 20 μM for 30 min), potassium depletion (K+ dep; 2 h) or treated with pneumolysin (PL) alone. Untreated controls and pneumolysin-treated cells were not primed with LPS. Cells were treated with or without 5 mM glycine (Gly), necrosulfonamide (NSA) (5 μM), or PL (0.5 μg ml−1). a Immunoblot analysis of released HMGB1 and cleaved IL-1β (p17) into the cell culture supernatant. PL causes release of HMGB1, but not IL-1 β processing. b LDH present in the extracellular media was quantified as a measure of cytotoxicity, with LPS + Ng and PL causing similar amounts of cell lysis. Data shown from n = 3 independent experiments. c Immunofluorescence of control and PL-treated BMDM. HMGB1 (green) and nuclei (DAPI; blue) indicate loss of nuclear HMGB1 in response to PL treatment. 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.