a BMDM were untreated or primed with LPS for 5 h (0.5 μg/ml) and then treated with or without nigericin (Ng; 20 μM) for the last 30 min. HMGB1 present in the extracellular media (supernatant) was quantified by ELISA. n = 3 (mean ± SEM). LPS treatment alone did not result in secretion of HMGB1 into the extracellular media. *p = 0.0009, n.s. not significant as determined by ANOVA with Tukey’s multiple comparison correction, two-sided. b BMDM were untreated, or treated with LPS for 5 h (0.5 μg ml−1. Cells were lysed and immunoblotted for HMGB1 and GAPDH. Note that HMGB1 expression does not change in response to LPS treatment. c, d BMDM were primed with LPS for 5 h (0.5 μg ml−1). The cells were fixed and immunostained for HMGB1 and LAMP1 or Rab7 and imaged by spinning disk confocal microscopy. Collapsed (Extended focus) images at ×63 magnification, size bar 20 μm. HMGB1 and LAMP1 or Rab7 do not colocalize in unstimulated or LPS-treated cells.