Relationship between caveolae and uPAR distribution in ECFC cell membrane. (A) Morphologic profile of ECFC colonies appeared after 2–3 weeks of culture (left panel). Capillary-morphogenesis in vitro at 6 and 24 hours (middle and right panel); (B) Caveolin-1 distribution. Cell lysates were layered on Optiprep solution and centrifuged as described in “Methods” to separate lipid rafts (fractions 2-6) from (fractions 7-9) in control conditions and after treatment with β-MCD (5mM for 20 minutes). Fractions obtained were probed by Western blotting with specific anti–caveolin-1 and anti-integrin β1 antibodies, as caveolar raft and nonraft marker respectively. Data are representative of 5 independent experiments. (C) β-MCD-treatment: right panel shows the β-MCD-induced cholesterol depletion. Evaluation of cholesterol amount was performed as previously described²² ; left panel shows vitality of the β-MCD-treated cells determined by trypan blue dye exclusion test. *P < .05, significantly different from control; (D) uPAR distribution: control and β-MCD–treated cells were subjected to Optiprep gradient separation and caveolar raft (CR) and nonraft (NR) fractions were analyzed by Western blotting with anti-uPAR antibody M2, which reveals both the full length and truncated uPAR. Numbers on the right indicates molecular weights expressed in kilodaltons. Data are representative of 5 independent experiments performed with clones expressing different levels of uPAR; (E) VEGF treatment: expression of uPAR and caveolin-1. Left panels: PCR of uPAR and caveolin-1 cDNA in untreated (control; lane 1) and VEGF-treated (lane 2) ECFCs. Numbers on the left indicate the size of PCR products in base pairs (bp). GAPDH: glyceraldehyde-3-phosphate dehydrogenase used as housekeeping gene. Right panels show Western blotting for uPAR and caveolin-1. Numbers on the right indicate molecular weights expressed in kilodaltons; (F) uPAR distribution in control and in VEGF-treated ECFCs. Control and VEGF-treated cells were subjected to Optiprep gradient separation: caveolar raft (CR) and nonraft (NR) fractions were collected and analyzed by Western blotting with anti-uPAR antibody M2. Numbers on the right indicate molecular weights expressed in kilodaltons; (G) silencing of uPAR gene using anti-uPAR aODNs: cell cultures were treated with aODN for 4 days in the presence of the cationic phospholipid DOTAP. A scrambled ODN (sODN) was used as a negative control. Top panels: PCR of ODNs-treated cells (on the left); Western blotting of ODNs-treated cells (α-tubulin was used as a loading control; on the right). Bottom panels: distribution of caveolin in Optiprep-prepared caveolar rafts after ODNs treatment. Data shown in panels E, F, and G are representative of 5 independent experiments.