Figure 2

Angiogenic effects of anosmin-1 in endothelial cells. (a) Transwell cell migration assay. HUVECs or bEnd3 cells were seeded in the upper compartment, and were treated with the indicated concentrations of anosmin-1, VEGF-A, both 7.5 nM anosmin-1 and 1 nM VEGF-A (Anosmin-1 + VEGF-A), or without reagents (0 nM). The cells that migrated through the Transwell membrane and attached on the underside of the membrane were counted in five different microscopic fields and quantified. (b) Cell proliferation assay. Starved HUVECs or bEnd3 cells were treated with the indicated concentrations of anosmin-1, VEGF-A, both 7.5 nM anosmin-1 and 1 nM VEGF-A (Anosmin-1 + VEGF-A), or without reagents (0 nM). After the incubation, the number of cells in the culture dishes was counted. (c) Representative images of the 2D tube formation assay. Cells were seeded on a 24-well plate coated with Matrigel, and then, 7.5 nM anosmin-1, 1 nM VEGF-A or both 7.5 nM anosmin-1 and 1 nM VEGF-A (Anosmin-1 + VEGF-A) were added. As a negative control (Control), cells were incubated without reagents. After the incubation and fixation, the formed tubes were observed by light microscopy. Scale bar: 200 μm. (d) Summary graphs of (c). Total length of the formed tubes was measured. *<0.05 and **P < 0.01 vs. without treatment (0 nM); ^†P < 0.05 and ^††P < 0.01 vs. 7.5 nM anosmin-1; ^§P < 0.05 and ^§§P < 0.01 vs. 1 nM VEGF-A.