Figure 3. CD93 or DG knockdown impairs EC function.

HUVEC were infected with a lentiviral vector expressing unrelated (unr), or CD93 (clones 85 or 86), or DG (clones C7 or C10) shRNAs. Not infected ECs were also analyzed (HUVEC). (A) Cell viability assay performed at the indicated time points on infected HUVEC plated in 96-well plates and grown in complete medium. The optical density values were expressed as relative cell viability. (B) ECs were infected, plated in 24-well plates and grown in complete medium. Cell number was evaluated by using a hemocytometer at the indicated time points. (C) Migration assay on infected HUVEC. Cells were grown in growth factor-depleted culture medium and plated in a Boyden chamber. Chemotaxis was stimulated with 10 ng/ml VEGF (Sigma-Aldrich). Migratory cells were stained and counted under a light microscope. (D and E) Wound healing assays of HUVEC infected as indicated. Cell monolayers were wounded with a sterile pipette tip, washed, and grown in complete medium. Cells were observed under a light microscope and photographed at 0 and 8 h. Representative experiments are shown (original magnification, x100). (F) HUVEC infected as indicated were grown in complete medium on Matrigel and the formation of capillary-like structures was observed 20 h after seeding. A representative experiment is shown (original magnification, ×100). (G) Quantification of tube length was performed based on the results shown in panel F. Results were expressed as means ± SD of four different fields randomly chosen from each group. All data represent the means ± SD of three-five independent experiments.