Figure 1.

Functionality of small hairpin RNAs (shRNAs) in vitro. (a) Inhibitory effects of shRNAs against Src kinase were analysed in human umbilical vein endothelial cells (HUVECs) on a protein level by western blotting. β-Actin was used as a normalization control for sample loading. Effect of Src inhibition on the cell secretion of matrix metalloproteinase 2 (MMP-2) was analyzed from +/− EGF-stimulated U118MG cell supernatants. Quantifications of western blots are shown below each lane. (b) Src inhibition in HUVECs was also measured on mRNA level by quantitative real-time reverse transcription (RT)-PCR. Target gene expression was normalized to GAPDH mRNA expression. **P < 0.01 versus nontransduced cells. (c) The effect of vascular endothelial growth factor (VEGF)-stimulation (50 ng/ml) on cell viability was measured by MTT assay in cells transduced with shRNAs. *P < 0.05; ***P < 0.001 versus nonstimulated cells. (d) To mimic angiogenesis in vitro, transduced cells were plated on growth factor reduced Matrigel and monitored over a period of several hours. Light and fluorescence microscopic pictures were taken. Representative pictures are from 6 hours timepoint. 40× magnification, scale = 1 mm. +, stimulated; −, nonstimulated; Ctrl, transduced with a control vector expressing shRNA against luciferase; EGF, epidermal growth factor; NT, nontransduced; sh1, shRNA sequence 1 against Src; sh2, shRNA sequence 2 against Src. Error bars = SEM.