Figure 4

Gene knockdown of VEGFR-2, NP1 and NP2 attenuates VEGF-mediated cell survival. VEGFR siRNA was carried out in A549 (A) and SKMES1 (B) lung cancer cells over 24, 48 and 72 h and protein expression was examined by Western blot analysis. NSCLC cells were transfected with siNP1 (100 nM), siNP2 (100 nM), anti-KDR (10 μg/ml) antibody alone, and in combination, for 48 h. While an IgG isotype antibody was used as a control for antibody specificity for KDR, proliferation in response to each siRNA was measured relative to a scrambled control siRNA for siNP1 and siNP2. Exogenous recombinant VEGF (100 ng/ml) was added for a further 24 h following receptor knockdown/blockade, after which time, cell proliferation was measured (C) (A549 cells, *p < 0.001, control vs siNP1, siNP2, siCombo, anti-KDR; ^#p < 0.05, control vs VEGF; ^$p < 0.05, siNP1 + VEGF, siNP2 + VEGF, siCombo + VEGF, anti-KDR + VEGF vs VEGF alone, n = 3). (SKMES1 cells, *p < 0.001, control vs siNP1, siNP1, siCombo, anti-KDR, VEGF alone; ^$p < 0.05, siNP1 + VEGF, siNP2 + VEGF, siCombo + VEGF vs VEGF alone; *p < 0.05, anti-KDR + VEGF vs VEGF alone, n = 3). Data are expressed as the mean ± SEM from three independent experiments. Statistical analysis was carried out by ANOVA using the Bonferroni multiple comparison post test.