E2-2 blocks EC activation. (A) E2-2 inhibits the formation of cord-like structures on the Matrigel. Forty hours after adenoviral infection, HUVECs were seeded on the Matrigel. Ninety minutes later, images were recorded every 15 minutes by time-lapse microscopy (supplemental Figure 4A-B). The images at 0-, 2-, and 4-hour time points are shown. Bottom panel: Expression of Myc-E2-2. Samples were visualized using a conventional microscope (Axiovert 200M; Carl Zeiss) with a 5×/0.12 dry objective lenses (Carl Zeiss). Images were acquired with AxioCam MRm 60-C1 (Carl Zeiss) and processed with the AxioVision Rel 4.4 (Carl Zeiss) and Adobe Photoshop 7.0.1 software (Adobe). (B) Id1 rescues E2-2–mediated inhibition of cell migration. After adenoviral infection, HUVECs were seeded on the upper membrane of the Boyden chamber. VEGF (50 ng/mL) was added to the lower chamber. After 6 hours, cells were stained with crystal violet, and the number of transmigrated cells was counted. Adenoviruses expressing LacZ or GFP were used as controls. Values represent the mean plus or minus SD (n = 3). Bottom panels: Myc-E2-2 and Flag-Id1 expression levels. Significant difference was calculated by the Student t test. (C) shE2-2 enhances VEGF-induced effect on HUVEC migration. Lentiviruses expressing GFP alone, shE2-2#1, or shE2-2#4 were infected in HUVECs. After sorting lentivirus-infected cells using GFP as a marker, sorted HUVECs were used for migration assay as described in panel B. Bottom panels: Expressions of E2-2 and β-actin by RT-PCR. Values represent the mean plus or minus SD (n = 3). Significant difference was calculated by the Student t test.