FIG. 6.

EBNA3C and Nm23-H1 can rescue HUVECs from antiangiogenic activity of Necdin. 293T cells were transfected with Necdin and vector control, EBNA3C, or Nm23-H1 expression constructs. The cells were allowed to grow for 48 h, after which the supernatant medium was harvested and stored at 4°C until used as CM. Each well of a 24-well plate was coated with 150 μl of ice-cold Matrigel (BD Biosciences), which was incubated at 37°C for about 30 min. A total of 5 × 10⁴ HUVECs were seeded on each well and grown in 500 μl of CM with 10% FBS. After 16 h of incubation, the cells were photographed to detect the formation of tubes structure by HUVECs. Tube structures were formed with the vector control (a) but not with Necdin (b). CM harvested from cells expressing EBNA3C with Necdin and from cells expressing EBNA3C together with Nm23-H1 and Necdin did not show an antiangiogenic effect (c and e), but medium from cells expressing Nm23-H1 with Necdin did show some antiangiogenic activity (d).