Figure 8. HMGB1 induces leukocyte adhesion to HRMECs and the expressions of cell adhesion molecules. A: The dose-dependent effects of rh HMGB1 on leukocyte adhesion. HRMEC monolayers in 96-well plates were treated with different doses of HMGB1 for 24 h and then LeukoTracker-labeled human leukocytes were allowed to attach to HRMEC for 30 min. Adherent cells were lysed and quantified as described in the method section. Each group has at least three wells. B: TNF-α (1 ng/ml; *p<0.05; Mann–Whitney U test) and HMGB1 (100 ng/ml; #p<0.05; Mann–Whitney U test) induced human leukocyte adhesion to HRMEC monolayers. The bar graph represents three independent experiments (control 10 wells, TNF-α 6 wells, HMGB1 10 wells). RFU = relative fluorescent unit. C: Effects of HMGB1 on the expressions of ICAM-1, VCAM-1, and VAP-1 on HRMECs. The protein expressions of ICAM-1, VCAM-1, and VAP-1 in treated and untreated (control) cell lysates were determined by western blot analysis. Western blots are representative of at least three independent experiments, each performed in triplicate, and bar graphs are representative of all three experiments. (*p<0.05; Mann-Whitney U test).