Fig. 1.

HMGB1 mediates important roles in HUVECs. A, Immunofluorescent staining (HMGB1, red; DAPI, blue; scale bars, 25 μm) for HUVECs exposed to hypoxia (1%) for 24 h. Photomicrographs are representative of three independent experiments. B, Representative capillary-like networks (tube formation, scale bars, 50 μm) of HUVECs with or without purified recombinant human HMGB1 treatment. C and D, Representative Western blot (C) and quantitative analysis (D) of HMGB1 in the cell media of HUVECs exposed to hypoxia. E, Total tube length from each of four randomly chosen fields was quantified using the image analysis software image. F and H, HMGB1 silencing of HUVECs. F, representative blots for HMGB1 silencing of HUVECs. H, HMGB1 siRNA silenced 60% of HUVEC HMGB1 protein expression. G, J, I, Tube formation and proliferation were tested after HMGB1 siRNA transfection. G, Representative capillary-like networks of HUVECs after HMGB1 silencing. (scale bars, 100 μm). and J, Quantifications of EC network formation in HUVECs with HMGB1 siRNA. I, the proliferation of HUVECs transfected with HMGB1 siRNA was decreased by 24%. Data are the average of triplicates from single experiments that were independently repeated 4–5 times. Band densities were normalized with that of β-actin. Comparisons were performed by using two-tailed unpaired Student's t-tests for D, E. Comparisons were performed by using one way ANOVA for H, I, J. (*P < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)