HLX does not affect the proliferation of endothelial cells, whereas migration is strongly reduced. (A) Proliferation assay: HUVECs were infected with HLX-encoding adenoviruses (Ad.HLX ▴) or control viruses (Ad.con ■) using an MOI of 8 or remained noninfected (without ♦). After 24 hours, 5 × 10³ cells were seeded per well into 96-well plates; 0, 1, 2, and 3 days after seeding the protein content was measured at OD₄₉₂ using the SRB assay_. The values display the increase in protein content over the indicated time period ± SD calculated from 5 wells each. One experiment of 3 with similar results is shown. (B) Migration assay: HUVECs were transduced with Ad.HLX or Ad.con with a MOI of 8 each or remained noninfected. Three days after infection, cells were seeded into a transwell system and the migration of the cells from the upper chamber toward the lower chamber containing medium with 50 ng of VEGF/mL or without VEGF was scored 4 hours after seeding of the cells. Cells migrated into the lower chamber were visualized by staining with DAPI. Five random microscopic fields per transwell were photographed at a magnification of 10-fold and the stained cells were counted. Values display the percentage of migrated cells in the individual samples in comparison to the VEGF-induced migration in samples containing noninfected cells arbitrarily set to 100%. Mean values were calculated from 2 independent experiments with triplicates each ± SD. Inhibition of migration by Ad.HLX was significant as calculated by t test (**P < .005, ***P < .001).