HMGB1 expression is impaired by inhibitor of the Atp binding cassette transporter ABC-1.

To characterize the secretion pathway of HMGB1, mononuclear cells were treated with 100 µM glyburide, ABC-1 inhibitor, in presence or absence of LPS (0.5 µg/ml). (A) After 20 h, cell surface-expressed HMGB1 was evaluated by FACS analysis in CB and PB cells. The values are shown as ratio between treated and untreated cells expressing HMGB1 (fold induction). The values are the mean ± SD of three experiments from different donors. Statistical analysis compared treated versus untreated cells (*P<0.05 paired Student's t test). (B) The culture medium of CB or PB cells, which have been analyzed in (A), has been evaluated by western blot analysis to detect its secretion. Western blot is representative of three independent experiments. (C) CB cells were stained with anti-HMGB1 antibody (green channel) and membrane-specific PKH26 red fluorescent dye and analyzed by confocal mycroscopy. HMGB1 expression was evaluated in untreated and glyburide treated CB cells (Top and bottom left panel) or LPS and LPS plus glyburide treated CB cells (Top and bottom right panel) after 20 h of activation.