H460 cells over-expressing NP1 promotes tumor growth in mice. H460 cells (NP1-negative) were transiently transfected with a NP1 over-expression vector, pcDNA3.1(-)-NP1, or empty vector control, pcDNA3.1(-) for 48 h and examined for its effect on cellular proliferation in the presence or absence of VEGF (100 ng/ml) (A) (*p < 0.05 vs EVC). Validation of NP1 over-expression in stably transfected H460 cells was carried out at the mRNA and protein levels using RT-PCR and Western blot analysis, respectively (B). The effect of VEGF stimulation on the proliferation of empty vector control cells and NP1 stably transfected cells were examined using the BrdU Cell Proliferation ELISA (C) (*p < 0.05, **p < 0.01). The response of NP1 over-expressing cells to VEGF blockade was examined using the BrdU Cell Proliferation ELISA following treatment with VEGF neutralizing antibodies (D) (*p < 0.05). In order to further confirm a role of the PI3-K and MAPK signaling pathways in VEGF-mediated NP1 survival signaling, protein expression of the downstream signaling proteins, phospho-Akt and phospho-Erk1/2, was examined by Western blot analysis (E) (*p < 0.05). Using an in vivo model, a tumor growth study was carried out using NP1 over-expressing H460 lung tumor cells in female nude mice. NP1 stably transfected H460 cells (3 × 106), or empty vector control cells, were injected subcutaneously on the left-hand side dorsal flank of each mouse (n = 8/group). Tumor volumes were recorded every 3-4 days for 24 days (F). From day 7 and up to day 24, by which time tumors had reached 2 cm3, lung tumor growth had increased significantly in mice injected with NP1 over-expressing cells (**p < 0.01; ***p < 0.001) compared to the much slower growing tumors observed in the control (EVC) group (G). Data are represented as the mean ± SEM from three independent experiments (A, C, D, and E). Statistical analysis for the in vitro analysis was carried out by ANOVA using the Bonferroni multiple comparison post test. For the xenograft study, a non-parametric Mann-Whitney Test was used.