Effect of AGF on growth and migration of endothelial cells. AGF induces endothelial cell migration, but does not affect the growth of endothelial cells. (A) First, 3 or 10 μg/mL AGF, 10 μg/mL ARP4, or 0.05 μg/mL VEGF was added to the lower chamber of the membrane. For each of these, 3 × 10⁴ mouse bEND3 cells per well were inoculated into the upper chamber of a transwell and were incubated for 4 hours at 37° C. The number of endothelial cells migrating from the upper to the lower chamber were counted. Columns represent mean values ± SD (n = 5). (B-C) Incorporation of ³H-labeled thymidine in HUVECs was evaluated as mitogenic activity. In panel B, confluent monolayers of HUVECs were made quiescent for 18 hours and then treated with various concentrations of VEGF. First, 1 μCi (0.037 MBq) ³H-labeled thymidine was added to each well. After a 4-hour incubation, mitogenic activity was assessed by measuring the uptake of ³H-labeled thymidine as counts per minute (cpm). Panel C shows the assessment of mitogenic activity of AGF for endothelial cells. Confluent monolayers of HUVECs were made quiescent for 18 hours and then treated with various concentrations of AGF with (•) or without (○) 10 ng/mL VEGF. The data are presented as mean values ± SD.