Figure 3.

In vitro characterizations of Scg3 as a novel vascular permeability and angiogenic factor. (A–C) Scg3 promotes endothelial permeability. Permeability assay with Transwell inserts is illustrated in A. 1 µg/ml Scg3, 100 ng/ml VEGF, or PBS was added to the bottom (B) or upper (C) chamber. After 24 h, media were collected from the top chamber and quantified for leaked FITC-dextran. n = 3 wells. (D) Endothelial proliferation assay with HUVECs in 48-well plates. Cell number in each well was quantified at 48 h. Scg3, 1 µg/ml; VEGF, 50 ng/ml. n = 4 wells. (E and F) Scg3 induces endothelial proliferation only at a high concentration. HUVECs (n = 4 wells; E) or HRMVECs (n = 6; F) were incubated with increasing concentrations of Scg3 in 96-well plates for 48 h. Cells in each well were quantified. (G–J) Tube formation assay with HUVECs. (G) Representative images of the tube formation. Scg3, 300 ng/ml; VEGF, 50 ng/ml. Bar, 20 µm. (H) Total tube length per viewing field. (I) Number of tubes per viewing field. (J) Number of branching points per viewing field. n = 4 fields. (K and L) Scg3 stimulates the migration of HRMVECs. (K) Representative images of cell migration. Bar, 100 µm. (L) The percentage of the denuded area covered by migrated cells within the original scratch was quantified. n = 3. Experiments were independently repeated three times. One representative experiment is presented. Data are ±SEM. One-way ANOVA was used.