FIGURE 3.

Silencing of the kdr gene impedes NRP-1 driven tube formation and related signaling in HDMEC. A and B, influence of addition of VEGF-A₁₆₅ (10 ng/ml) and Fc rNRP-1 (5 nm) on HDMEC tube formation on collagen substratum (white bar indicates 100 μm) on four pools of cells, where one comprised of untrasfected cells, second of the cells transfected with non-silencing siRNA (N.S. siRNA) and third and fourth of cells transfected by two different siRNAs against kdr gene. The tubes were photographed and analyzed as described under “Experimental Procedures.” The relative values depicting tubular morphogenesis were normalized versus control variant of the untransfected cells. Significant differences between the tested agonists and their respective controls were determined by Student's t test. Single asterisk (*) indicates that the p value is <0.01. C, Western blot of each pool of cells from a parallel experiment conducted on a layer of collagen. Samples were collected for analysis 10 min after addition of VEGF-A₁₆₅ (10 ng/ml), shNRP-1, and Fc rNRP-1 (10 nm). The band intensities were analyzed as described under “Experimental Procedures” and normalized versus the untransfected cell control.