FIGURE 6.

Inhibition of Matrigel^TM invasion and chemotaxis of capillary endothelial cells after tunicamycin treatment. A, synchronized culture of capillary endothelial cells either alone or after pretreating with tunicamycin (1 μg/ml) for 32 h were seeded in control and growth factor-reduced Matrigel^TM-coated transwell plates. B, cells were cultured in EMEM containing 2% fetal bovine serum in the upper chamber. Conditioned media from human breast cancer cells MCF-7 cultured in 10% fetal bovine serum along with VEGF (10 ng/ml) was used in the lower chamber as a chemo-attractant. After incubation for 24 h at 37 °C in a CO₂ incubator, the transwells were removed, cells passed through the membrane were fixed and stained with H&E. The invaded cells were quantified by counting in an optical microscope at 200× magnification and averaged after counting five fields per membrane. The histogram at the right is the quantification of cell invasion through Matrigel^TM (p < 0.001). C, image of chemotaxis of endothelial cells. Endothelial cells grown to confluence in a regular media containing 10% fetal bovine serum and switched to a media containing 0.2% fetal bovine serum for 6 h. The monolayers were scratched with a 10 μl pipette tip and cultured with or without VEGF (10 ng/ml), or tunicamycin (1 μg/ml), or tunicamycin (1 μg/ml) + VEGF₁₆₅ (10 ng/ml) for an additional 6 h. The migrated cells were quantified microscopically and the histogram at the right is the quantification of cell migration (averaging the position of the migrating cells at the wounding edges; p < 0.001); broken line indicated by →.