Figure 3
Biological effects of dimeric CMP-Ang1 in HUVECs.
(A,B) Confluent HUVECs were starved in serum-free medium and stimulated with either control buffer (CB), 500 ng/ml of CA1-1, CA1-2, CA1-3, GCN-Ang1, FC-Ang1, or COMP-Ang1 for 15 min. (A) Images of HUVECs stained for Tie2 (green) and nucleus (blue). Scale bars, 10 μm. (B) Quantification of the fluorescence intensity (FI) of Tie2 at cell junction. (C,D) HUVECs were incubated with serum-free medium containing CB or 500 ng/ml of Ang1 variants. (C) Phase-contrast photographs were taken at 48 hr after serum starvation. Scale bars, 100 μm. (D) Apoptotic cells were measured by TUNEL immunostaining and flow cytometry. (E,F) HUVECs were plated on MatrigelTM coated wells and were incubated with serum-free medium stimulated with CB or 500 ng/ml of Ang1 variants. (E) Phase-contrast photographs were taken at 7 hr after treatment. Scale bars, 100 μm. (F) Tube formation activities were measured by total tube length. (G,H) Migration activity assay was performed using a modified Boyden chamber assay. HUVECs were seeded in the upper layer of 8 μm-pore membrane. Serum-free medium containing 500 ng/ml of Ang1 variants was added in the bottom of the chamber. (G) The migrated cells were fixed and stained after 9 hr incubation. Scale bars, 100 μm. (H) The migrated cells on the bottom of membrane were counted. Each graph indicates the mean ± SD of three experiments. *p < 0.05 versus CB.
