mAb AA98 inhibits proliferation and migration of HUVECs and does not promote apoptosis. (A) HUVECs or A375 melanoma cells were incubated with mAb AA98 in culture medium at indicated concentrations for 48 hours followed by incubation with [³H]-thymidine for 4 hours. The inhibition of [³H]-thymidine incorporation by mAb AA98 (100 μg/mL) compared with control mIgG (100 μg/mL) showed that mAb AA98 had a significant inhibitory effect on the proliferation of HUVECs, whereas proliferation of A375 cells was not affected. The assays were performed 2 times in duplicate and the data represent the average of duplicate determinations from a single representative experiment. The range of the values for the duplicate determinations was less than 10%. (B) Migration of HUVECs toward VEGF/bFGF in the presence of 10 μg/mL mAb AA98 or control mIgG. The migration assay was allowed to proceed for 4 hours, and the number of cells reaching the lower chamber—side of the membrane were counted at high-power fields (original magnification, × 200). Graphic representation of migrated cells is shown as the mean ± SD of 10 fields on duplicated filters. (C) mAb AA98 does not promote apoptosis of proliferating HUVECs or A375 cells. Apoptosis assays were performed with cultured HUVECs and A375 cells in the presence or absence (untreated) of antibodies mAb AA98 or mIgG (negative control). Cells cultured in the presence of the apoptosis-promoting agent doxorubicin (DOX) at 1 μM served as positive apoptosis control. Concentrations of antibodies are indicated on the x-axis. Absorbance on the y-axis is directly proportional to the extent of apoptosis. Each point represents the mean of triplicate determinations; error bars, ± SD.