Figure 5.

Decreases in cell migration, survival, angiogenesis, and invasion in human Ewing’s sarcoma SK-N-MC and RD-ES cell lines. Treatments: scrambled shRNA plasmid (0.5 μg/ml) transfection for 72 h, EWS shRNA plasmid (0.5 μg/ml) transfection for 72 h, 100 μM TFL treatment for 24 h, and EWS shRNA palsmid (0.5 μg/ml) transfection for 48 h + 100 μM TFL treatment for last 24 h. (A) Cell migration assay. Cells (1 × 10⁵) were seeded for migration through the matrigel coated membrane of tranwell insert following incubation at 37°C in presence of 5% CO₂ and full-humidity for 48 h. The membranes were collected, stained, and photographed under the light microscope. (B) Quantitation of matrigel invaded cells underneath the membrane. Mean values (n = 3) were shown and significant difference between two values was indicated by *P < 0.05 or **P < 0.01 (where monotherapy or combination therapy was compared with scrambled shRNA) and ^#P < 0.01 (where combination therapy was compared with monotherapy). (C) Western blotting to examine decreases in expression of specific molecules involved in cell survival (p-Akt and NF-κB), angiogenesis (VEGF and b-FGF), and invasion (MMP-2 and MMP-9).