Fig. 4

Inhibition by D16F7 of VEGF-A and PlGF-induced phosphorylation of VEGFR-1 at Tyr 1213 in GBM cells over-expressing VEGFR-1. a VEGFR-1 mRNA levels in U87-derived clones transfected with control (U87-CTR6) or VEGFR-1 expressing (U87-MF1 and U87-MF24) vectors was analyzed by RT-PCR. Amplified products were separated on 1% agarose gels and results are representative of one out of two different experiments giving comparable results. b VEGFR-1 protein levels in U87-derived clones transfected with control or VEGFR-1 expressing vectors were analyzed by Western blotting. Numbers below immunoblot lanes indicate VEGFR-1/β-actin optical density (O.D.) ratios. c Western blotting of total or phosphorylated VEGFR-1 (pVEGFR-1) at tyrosine 1213 and total or phosphorylated Erk1/2 (pErk) in untreated or D16F7 (1 or 10 μg/ml) pre-treated U87-MF24 cells in response to PlGF or VEGF-A. Histograms represent the densitometric quantification of band intensities in the corresponding immunoblots, expressed as pVEGFR-1/VEGFR-1 ratio relative to untreated control, after normalization for β-actin expression. Normalized pVEGFR-1/VEGFR-1 or pErk/Erk protein ratio in untreated cells was considered equal to 1. d Histogram represents the mean ± SD percentage inhibition values of PlGF or VEGF-A-induced VEGFR-1 phosphorylation or Erk1/2 phosphorylation in U87-MF24 cells after treatment with 1 and 10 μg/ml D16F7, calculated from immunoblot densitometric analysis of three independent experiments. e For spheroid invasion assay U87-MF24 cells were embedded in matrigel in the absence or presence of D16F7 (10 μg/ml) and PlGF (50 ng/ml). Representative pictures of spheroids taken at 24, 48 and 72 h after embedding cells in matrigel (40× magnification) are shown; NS, non-stimulated cells. Relative invasion was quantified as described in Fig. 1d legend. Data are expressed as mean ± SD (n = 6–10) and results of statistical analysis were as follows: PlGF vs NS, PlGF vs D16F7 or PlGF vs PlGF + D16F7, p < 0.001 (***) at 48 and 72 h. Differences between NS and PlGF + D16F7 were not significant