Caveolin-1 silencing. Effects on invasion, capillary morphogenesis and VEGF signaling. (A) Left panels: PCR and Western blotting of caveolin-1 silencing in ECFCs. Numbers on the left: size of PCR products (bp); numbers on the right: molecular weights expressed in kilodaltons. Right panel: Western blotting of uPAR distribution between caveolar raft (CR) and nonraft (NR) fractions under control conditions (scrambled-sequence siRNA) and after caveolin-1 silencing (Cav-1 siRNA). uPAR was revealed with M2 antibody. Numbers on the right: molecular weights expressed in kilodaltons. Results were similar to those Figure 1D, obtained with β-MCD. (B) Effects of caveolin-1 silencing on basal and VEGF-stimulated ECFC Matrigel invasion (top panels) and ECFC capillary morphogenesis (bottom panels). For image acquisition and quantification, refer to the legends of Figures 1 and 3. Quantification is shown by histograms on the right: values refer to 3 experiments performed in triplicate in 3 different ECFC cell lines. Data are expressed as mean value ± SD. *P < .05, significantly different from control. (C) Western blotting with anti-pERK1/2 antibodies in the experimental conditions reported in the bottom captions (control conditions, β-MCD treatment, caveolin-1 silencing, and VEGF stimulation), showing that both β-MCD treatment and caveolin-1 silencing inhibited VEGF-dependent ERK1/2 phosphorylation. ERK1/2 indicates loading control obtained with anti-total ERK1/2 antibodies. Numbers on the right: molecular weight expressed in kilodaltons. Histograms on the bottom show quantification of Western blotting experiments (as densitometric units) and refer to 3 experiments performed in triplicate in 3 different ECFC lines. Data are expressed as mean value ± SD. *P < .05, significantly different from control.