shRNA gene transfection downregulates gene expression in vivo. Established LS-174T tumors (~100 mm3 in volume) were injected with either control vector (A, C), Del1-shRNA (B) or VEGF-shRNA (D) expression plasmids. Illustrated are representative tumor sections prepared 4 days following gene transfer, stained brown with Abs against Del1 (A, B) or VEGF (C, D). Tumoral expression of Del1 (E) or VEGF (F) was detected by Western blot analysis. Blots of homogenates from tumors injected with control vector (lane 1) and shRNA plasmid (lane 2) were stained with Abs against Del1 (upper row) (E), or VEGF (upper row) (F), and an anti-β-actin Ab (lower row). The density of each band was measured and normalized to that of β-actin. *Indicates a significant difference at P < 0.05 in the band intensities of Del1 or VEGF between control vector transfected groups.