Inhibition of adhesion of WM266 on different extracellular matrix coated plates (A).

<p>Cells were pre-incubated with peptides (50 µM) or anti-αvβ3 antibody (10 µg/mL) for 30 min at 4°C and then seeded on extracellular matrix pre-coated plates. Cell adhesion was evaluated after 1 h of incubation using crystal violet reagent. The results are presented as the percentage of adherent cells respect to the control (untreated cells) and are expressed as means ± SE of three independent experiments performed in triplicate. Statistical significance was analyzed using Student's t test, unpaired, two-sided (*p<0.05). RGDechi-hCit dose-effect on WM266 cell adhesion (B). Cells were pre-incubated with increasing concentrations of RGDechi-hCit for 30 min at 4°C and then seeded on vitronectin or fibronectin (10 µg/mL) pre-coated plates at 37°C. The cell adhesion was evaluated after 1 h of incubation using crystal violet reagent. The results are presented as the percentage of adherent cells respect to the control (untreated cells) and are expressed as mean ± DS from three independent experiments performed in triplicate.</p

Detachment of WM266 cells from vitronectin coated plates.

<p>Cells were plated onto vitronectin (10 µg/mL) pre-coated wells and were allowed to adhere for 1 h. Peptides (50 µM) or anti-αvβ3 antibody (10 µg/mL) were added and the incubation was continued for 2 h. Cell adhesion was determined by crystal violet assay. Results are presented as percentage of adherent cells respect to the control (untreated cells) and are expressed as means ± SE of three independent experiments performed in triplicate. Statistical significance was analyzed using Student's t test, unpaired, two-sided (* p<0.05).</p

Merge of confocal and transmitted light images of peptide cellular uptake.

<p>WM266 cells after 30 min incubation with 50 µM RGDechi-hCit (A) and scrambled peptide(B). HeLa cells after 30 minute incubation with 50 µM RGDechi-hCit (C). Green fluorescence indicates peptides. Magnification bar: 20 µm.</p

Cytotoxicity assay.

<p>WM266 were treated with starvation conditions (4% FCS) for 4 h and then peptides at different concentrations were added and incubated for 24 h. The proliferation was evaluated using crystal violet assay. The results are presented as the percentage of proliferating cells respect to the control (untreated cells) and are expressed as means ± SE of three independent experiments performed in triplicate. Statistical significance was analyzed using Student's t test, unpaired, two-sided (* p<0.05, **p<0.01).</p

RGDechi cellular uptake inhibition experiments.

<p>A–F, Confocal microscope images of WM266 cells after 30 minutes of incubation with 50 µM RGDechi. A and D control non-treated cells; B and E cells treated with 0.45 M sucrose; C and F cells treated with 5 µg/ml filipin. A–C fluorescence images; D–F merge of fluorescence and transmitted light images. Magnification bar 50 µm. G, quantitative analysis of RGDechi cellular uptake by spectrofluorimeter measurements. Data are expressed as mean ± standard deviation (* p<0.05).</p

Co-localization areas (white arrows) of RGDechi-hCit peptide (green) with αvβ3 integrin (red)(A), caveolin 1 (red)(B), clathrin (red)(C) and LAMP 2 (red)(D) in WM266 cells after 30 minute incubation.

<p>(A, C, E, G) fluorescence images; (B, D, F, H) transmitted and fluorescence image overlapping. Magnification bar: 20 µm.</p

Analysis of expression of αvβ3 integrin in WM266 and HeLa cells by flow cytometry.

<p>Pre-confluence cells (WM266 cells (A), HeLa cells(B)) were detached by 0.1 mM EDTA in PBS and incubated with anti αvβ3 antibody (green curve) or isotype (black curve) and then with secondary antibody FITC conjugate.</p

Apoptosis analyses with annexin V-FITC/PI double staining on WM266 cells.

<p>Untreated cells (A); cilengitide treated cells (B); RGDechi-hCit treated cells (C). Upper left quadrant: necrotic cells; upper right: advanced apoptotic cells; lower left: viable cells; lower right: early apoptotic cells. These pictures are representative of three independent experiments.</p