Figure 1.

Ery5 diminishes angiogenesis in HUVECs and PC-3 cells. (a) Ery5-attenuated HUVEC proliferation. HUVECs were plated in 96-well plates, treated with various concentrations of Ery5 for 24 h. MTT was added 3 h before the termination of the experiment; the procedure of the assay is discussed in Materials and Methods. Data are mean±S.D. of three similar experiments; statistical analysis was done by using the Bonferroni method and P-value<0.05 was considered to be significant with ***P<0.001. (b and c) Ery5-inhibited colony formation in PC-3 cells. PC-3 cells were treated with different concentrations of Ery5 for 5 days; the procedure of the assay is described in Materials and Methods. (d) Ery5-inhibited tube formation in HUVECs. For tube formation assay, 50 μl of extracellular matrix was transferred to each well of a precooled 96-well tissue culture plate. Then the plate was incubated at 37 °C for at least 1 h to allow the matrix solution to solidify. HUVECs were added to each well and were allowed to attach overnight. Cells were treated with 1 μM of sunitinib and 5, 10 and 20 μM of Ery5 for 24 h. The tube formation was observed under an inverted light microscope at × 10 magnification using an inverted microscope equipped with digital camera (Olympus Imaging Corp.). Ares of formed tubes were measured as described in Materials and Methods. (e and f) Ery5-inhibited migration in HUVECs and PC-3 cells. HUVECs and PC-3 cells were plated in six-well plates. A wound was given with a sterile tip. Ery5 (10 μM) was added for 6, 12 and 24 h. The procedure for the assay is discussed in Materials and Methods. (g) Ery5-attenuated angiogenesis in ex vivo model. Ex vivo angiogenesis inhibition was analyzed through aortic arch ring formation assay. The procedure of the experiment is described in Materials and Methods. (h and i) Ery5 attenuated the expression of angiogenic proteins in PC-3 and HUVECs. Cells (1 × 10⁶) were plated in 60 mm dishes, cells were allowed to attach and grow and were incubated with 10 μM of Ery5 for 6, 12 and 24 h. Cells were lysed with RIPA buffer, proteins were separated on SDS-PAGE, transferred to PVDF membranes and probed for different antibodies as described in Materials and Methods