Fig. 3

HMGB1 release requires live Eh Gal-lectin-mediated contact with macrophages. a THP-1 cells were stimulated for 30 and 60 min with live Eh (20:1 ratio), secreted proteins (SPs, 50 μg) from Eh, whole lysates of Eh, and cytoplasmic and membrane components of an equal amount of Eh. Equal amounts of cell culture supernatant (SN) samples were loaded onto the SDS-PAGE gel for HMGB1 detection. b THP-1 cells were stimulated up to 16 h with LPS (100 ng/ml) and Eh sub-cellular fractions and secretion of HMGB1 protein was assessed. c Eh was pre-treated for 5 min with 55 mM d-galactose before incubation with macrophages. THP-1 cells were stimulated with either galactose-treated Eh or Eh only for 10 min to assess HMGB1 secretion. Densitometric analysis showed significant inhibition of HMGB1 secretion following galactose treatment. d THP-cells were treated with native Eh Gal-lectin (500 ng/ml) or LPS (100 ng/ml) for different time points (1–4 h) and HMGB1 secretion was determined by western blot. Data are from three independent experiments and statistical significance was determined by t test ***P < 0.001. Bar represent mean ± SEM.