Figure 5.

Evaluation of angiogenic response, cell proliferation and migration after silencing UGDH and EPI treatment. MDA-MB-231 cells were transfected with a specific siRNA against UGDH gene (siUGDH) using a random sequence siRNA as the negative control (siSCR). After 24h, 1 µM EPI (1 EPI) was added to complete 48 h of incubation. VEGF (A) and EGF (B) expressions were obtained by real-time quantitative PCR using SYBR Green. Results were normalized using β-actin as the reference gene and all determinations were performed as duplicates. Levels of secreted VEGF in cell supernatants (C) and intracellular levels of FGF-2 in cell lysates (D) were measured by ELISA. Total protein was extracted and the expression of β-catenin (E) and p-AKT (F) were detected by Western blot. The images show the most representative of the three independent experiments. To describe cell migration ability, consistently shaped wounds were made during transfection and EPI treatment. The experiment evaluated the same coordinates of each photo at different time points in order to evaluate migration ability. Three images were captured at 0 h, 4, 8 and 22 h at the same coordinates. The gap sizes of the wounds were measured and analyzed using ImageJ software. The results were shown as free area of the wound, which is inversely proportional to the migration ability of the cells. The results were expressed as the decrease in the initial area of the wound, considering as 100% the area at time 0 (G). Micrographs show the most representative of three independent experiments (H). n = 3, as mean ± SEM. * p < 0.05 ** p < 0.01 *** p < 0.001.