Egr-3 is required for VEGF-mediated induction of proliferation, migration, and tube formation of primary human endothelial cells. (A) Proliferation assay. HUVECs (105) were transfected with si-Control, si-Egr-3 oligo1, or si-Egr-3 oligo2. Cells were serum-starved for 18 hours, incubated in the presence of 50 ng/mL VEGF for 48 hours, and subsequently enumerated. The results show the mean ± SD derived from 6 independent experiments. *P < .01; **P < .04 compared with si-Control. (B) Migration assay. HUVECs were transfected with si-Control, si-Egr-3 oligo1, or si-Egr-3 oligo2; labeled with PKH2; serum-starved; and plated in upper layer of a Transwell. A total of 50 ng of VEGF (or vehicle) was added to the lower chamber. After 24 hours' incubation, migrated cells were detected by the use of a fluorescent microscope. The number of migrated cells (green) was quantified with image analysis software. Means ± SD were derived from 3 independent experiments, each performed in triplicate. *P < .01 compared with si-Control plus VEGF. (C) Scratch wound assay. Confluent HUVECs transfected with si-Control, si-Egr-3 oligo1, or si-Egr-3 oligo2 were scratched with the use of a 1-mm fine tip. After 24 hours of VEGF treatment, the number of cells migrating into the scratched area was counted. Red lines correspond to the borders of the scratched area. The graph shows mean ± SD of migrated cells derived from 4 independent experiments, each performed in triplicate. *P < .01, compared with si-Control. (D) Tube-formation assay. HUVECs were transfected with si-Control si-Egr-3 oligo1 or si-Egr-3 oligo2, labeled with PKH26, serum-starved, and grown on a collagen gel in the presence or absence of 50 ng/mL VEGF. Cells were observed under fluorescence (top) or bright field (bottom). White bar indicates 100 μm. The mean ± SD of total tube length was calculated with image analyzer from 3 independent experiments performed in triplicate (bottom bar graph). *P < .02 compared the activity from si-Control plus VEGF.
