Figure 3

PI3K and MAPK signaling pathways are stimulated by VEGF. A549 cells were treated with recombinant human VEGF (100 ng/ml), VEGF neutralizing antibodies (1 μg/ml) or both combined. Phospho-Akt and phospho-Erk1/2 expression (A) and localization (B) were examined by high content screening (HCS) and confocal microscopy, respectively. Expression of the phosphorylated proteins, pAkt and pErk1/2, were quantified using IN Cell Analyzer 1000 Software (phospho-Akt; *p < 0.05 untreated vs anti-VEGF, anti-VEGF vs combined, ^$p < 0.01 anti-VEGF vs rVEGF; phospho-Erk1/2; ^$p < 0.01 untreated vs anti-VEGF, anti-VEGF vs combined, ^$#p < 0.001 untreated vs rVEGF, anti-VEGF vs rVEGF, anti-VEGF vs combined, n = 3). Data are expressed as the mean ± SEM. Statistical analysis was carried out by ANOVA using the Bonferroni multiple comparison post test. Localization and expression levels of phospho-Akt and p44/p42 MAPK (Erk1/2) proteins were examined (×60 magnification) using a Zeiss LSM 510 laser scanning confocal microscope. Representative cells showing green fluorescence are indicative of phosphorylated Akt, while cell nuclei are stained blue. PI3-K and MAPK signaling proteins were examined by Western blot in response to siVEGF (C). VEGF mRNA expression was also assessed by RT-PCR to confirm knockdown of VEGF. A549 cells were treated with siRNA to VEGF (100nM) or a scrambled siRNA control for 48 h, after which time, cell proliferation was measured (D) (*p < 0.05, $p < 0.01, ^#p < 0.001, n = 3). Akt and MAPK (Erk1/2) phosphorylation was also examined in response to siVEGF (E). Data are represented as the mean ± SEM. Statistical analysis was carried out by ANOVA using the Bonferroni multiple comparison post test (^#p < 0.05, *p < 0.01, ^$p < 0.001, n = 3).