Figure 5.

Role of EPHA2 in angiogenesis in vitro (a–d) and in vivo (e–h). (a) Representative micrographs of the in vitro capillary-like structures formed by human umbilical vein endothelial cells (HUVECs), upon treatment with conditioned medium from untreated MKN74 cells (cells), MKN74 cells treated with a nonsilencing siRNA (siNS) as a negative control, or with an siRNA for the EPHA2 (siEPHA2) 5 h post-seeding in Matrigel-coated wells. (b) Corresponding automatic analysis using WimTube software (scale bar: 100 µm; original magnification: ×100) with the quantification of the number of tubes (c) and branching points (d) per microscopic field from 3 independent experiments. (e) Representative photomicrographs of the in vivo chicken embryo chorioallantoic membrane (CAM), depicting new blood vessel formation induced by untreated MKN74 cells (cells), MKN74 cells transfected with a nonsilencing siRNA negative control (siNS), or with an siRNA against the EPHA2 (siEPHA2). Cells were inoculated on top of the CAM inside a 5 mm silicon ring under sterile conditions for 3 days (scale bar: 1 mm; original magnification: 20×). (f) Representative immunohistochemistry of the CAM paraffin sections stained with EPHA2 antibody (scale bar: 50 µm; original magnification: 200×). (g) Quantification of the number of new vessels radially formed toward the inoculation area as a measure of the angiogenic potential of the inoculated cells. Data regarding 15 fertilized eggs per condition are depicted on the box plot graph. (h) Representative Western blot of EPHA2 and tubulin expression in MKN74 for the different experimental conditions at the end of the experiment, and the quantification for 3 independent experiments. Data are presented as mean ± SE. One-way ANOVA analysis followed by Tukey’s multi-comparison test: **** p < 0.0001; *** p < 0.001; ** p < 0.01; ns—not significant (p > 0.05).