Figure 8

Endostatin induces the activity of caspase-8. A) HDMEC were seeded on tissue culture dishes, and following removal of non-adherent cells by washing, were stimulated with either 50 ng/ml VEGF alone or in combination with 100, 500 or 2500 ng/ml recombinant endostatin for 48 h. Cells were collected and total protein lysates were generated. Western blots for procaspase-8 were performed as described in materials and methods. Blots were stripped and reprobed for tubulin as a loading control. B) HDMEC were seeded on tissue culture dishes, and following removal of non-adherent cells by washing, were stimulated with either regular media (unstimulated), or media containing 100, 500, or 2500 ng/ml endostatin, in the presence or absence of 50 ng/ml VEGF, or stimulated with anti-fas antibody as a positive control for caspase-8 activation. Cells were collected by trypsinization and cell pellets stored at -80°C. Subsequently, pellets were lysed and total protein concentration was determined. Caspase-8 activity was then detected using a commercially available fluorometric detection kit (Sigma, Oakville, ON) as described in the methods section. An additional sample was included using the anti-fas treated cell lysate in the presence of specific caspase-8 inhibitors to demonstrate the specificity of the assay.