Ated FCS (Hyclone, Logan, UT). Cells were cultured at 37 in a 5

Ated FCS (Hyclone, Logan, UT). Cells were cultured at 37 in a 5 CO2 atmosphere.Aguzzi et al. Molecular Cancer 2010, 9:84 http://www.molecular-cancer.com/content/9/1/Page 7 ofFigure 6 RGDS treatment activates caspase-3. A: SK-MEL-110 seeded on collagen-IV were incubated for 48 h with FGF-2 in the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/20703940s AA26-9 presence or in the absence of RGDS or RGES (500 g/ml). Top: Pro-caspase 3 cleavage and caspase-3 activation were observed by western blotting. Bottom: caspase-3 activation was confirmed by confocal microscopy using an anti-active caspase-3 antibody. B: Pre-treatment (2 h) of melanoma cells with Z-VAD-FMK (50 M), a general caspases inhibitor, abolished the RGDS anti-proliferative effect (p < 0.005 vs FGF-2 in the presence of Z-VAD-FMK). All these experiments were carried out three times in duplicate.Proliferation assay and apoptosisSK-MEL-110 were plated in 6-well plates (8 ?104 cells/ well) on collagen-IV (50 g/ml) and were grown for 24 h in complete medium in 5 CO2. Cells were then serumstarved for 24 h and subsequently treated with RGDS (Bachem, Bubendorf, Switzerland) or RGES as control (Sigma-Aldrich, St Louis, MO) (500 g/ml) dissolved in DMEM containing FGF-2 (Pierce-Endogen, Rockford, IL) for 48 h. Then, cells were photographed, harvested by trypsin-EDTA and counted using hemacytometer. In other experiments, cells were pre-treated with a general caspase inhibitor (Z-VAD-FMK) (50 M) (R D Systems, Minneapolis, MN) for 2 h before RGDS treatment. To analyze cell cycle and sub-G1-phase, cells were fixed in ice-cold 70 ethanol and stained with propidium iodide (PI) at 10 g/ml final concentration. Flow cytometry was performed on a Profile I flow cytometer (FACSCalibur, BD-Biosciences).Biotinylated-RGDS (bt-RGDS) internalization and interaction with recombinant proteinsTo investigate peptides internalization, cells were treated with the biotinylated-RGDS (bt-RGDS) or biotinylatedRGES (bt-RGES) (NeoMPS-SA, Strasbourg, France) as control for different time-points, stained with phycoerythrin conjugated-avidin and analyzed by FACS. In additional experiments cells grown on collagen-IV were serum-starved for 48 h and treated for 24 h with biotinalone or with different doses of bt-RGDS (10-50-100 g/ ml). In other experiments cells were treated with 50 g/ ml bt-RGDS with an excess of 1 mg/ml un-labeled RGDS as specific competitor. Cells were washed to eliminate btRGDS bound to the membranes and a cytoplasmic extract was prepared as previously reported [3]. Cytoplasmic lysates were spotted onto nitrocellulose, blocked with 5 milk in TPBS (0.1 Tween 20 in PBS) and incubated for 1 h at RT with a Vectastain ABC-peroxidase kit (Vector) followed by chemiluminescence reaction and exposure to Kodak film (Eastman Kodak). Interaction was quantified by densitometry (GS 710; Bio-Rad) and analyzed using the "Quantity one" software (Bio-Rad). In other experiments 40 g of growing cells cytoplasmic extract, or increasing doses of recombinant human survivin (0.3-0.9-1.5-3-5 g) (CP-Biotech, Sylvania, OH) or other recombinant proteins, as caspases-1 and -9, procaspase-9 (Alexis), fibronectin (0.9 g) (Becton-Dickinson, Bradford, MA) and BSA were spotted onto nitrocellulose, incubated for 4 h at RT with bt-RGDS (1 mg/ml) in the presence or absence of RGDS-excess (10 mg/ml) to measure the specific binding.Confocal microscopySK-MEL-110 seeded on coverslips coated with collagenIV and treated for 24 h with bt-RGDS (500 g/ml), were washed as previously reported to eliminate.