FIG. 7.

EBNA3C and Nm23-H1 rescue HeLa cells from Necdin-mediated anti-invasive effects. 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 37°C for about 30 min. A total of 5 × 10⁴ HeLa cells were seeded on each well and grown in the 500 μl CM with 10% FBS. After 16 h of incubation, the cells were photographed to detect the growth properties of HeLa cells. Necdin suppresses growth of HeLa cells (a, left panel), whereas Nm23-H1 (center panel) and EBNA3C (right panel) do not have any effect. Both EBNA3C and Nm23-H1 were able to rescue the cells from Necdin-mediated growth inhibition of HeLa cells (b). Necdin Δ144-184 (c) and Necdin Δ60-100 (e) did not show any suppression of HeLa cell growth, suggesting a role of these domains in Necdin-mediated growth inhibition. However, Necdin Δ191-222 did show partial inhibition of HeLa cell growth, and, more importantly, neither EBNA3C nor Nm23-H1 was able to rescue the cells from Necdin Δ191-222-mediated growth suppression (d).