FIGURE 4.

Decorin-mediated attenuation of HGF/Met signaling evokes potent angiostasis. A, immunoblot verification of Met protein depletion following transient transfection of cognate targeting siRNA (80 pm) (designated as siMet) in HeLa 48 h post-transfection. B, immunoblot analysis following positive verification of Met knockdown HeLa lysates probed for cellular VEGFA under conditions of control, decorin (24 h, 500 nm), siMet (80 pm), or combined treatment and appear as a quantification of cellular VEGFA fluorescent intensity after normalizing to β-actin. C, expression data gained from qPCR analysis of pertinent genes in the presence of siMet (80 pm). Verification of siRNA functionality is delineated in this graph for Met. D, HeLa lysates were immunoblotted for β-catenin and β-actin (as a loading control) under the influence of decorin (500 nm) for 24 h. E, HeLa cDNA libraries evaluated for CTNNB1 via qPCR under the same experimental conditions as in D. F, micrographs depicting high resolution digital images of mouse dorsal skin as it appears from the interior, 14 days after subcutaneous injection with either Matrigel (80 ml combined with ∼10⁶ MDA-231 GFP⁺ cells) and HGF (10 ng/ml) in the absence (F, left panel) or presence (F, right panel) of decorin proteoglycan (200 nm). Asterisk denotes Matrigel plug and tumor location. G, morphometric analysis of blood vessel density reported as vessel intersections per μm². Density was calculated by determining the average number of times the newly formed angiogenic vessels intersected a grid, representing a standard area of 1 mm², which were superimposed onto the images. The values represent the mean ± S.E. (n = 600). Bar = 4 mm. Immunoblot and Met knockdown experiments represent an average of at least two independent experiments performed in duplicate with quantification representing the average ± S.E. for cellular VEGFA. Gene expression data are representative of 2–3 independent trials reported as fold-change ± S.E. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).