Cell viability, adhesion, and aggregation assays

Cell viability was determined either using the WST-1 cell proliferation assay kit (Sigma-Aldrich) or by counting the number of live cells with a haemocytometer. Cell adhesion and cell aggregation assays were conducted as described previously^21,23. In short, cells were incubated in normal culture plastics without coating or pre-coated with specific ECM proteins, such as fibronectin (0.2 µg/ml), laminin (0.4 µg/ml), collagen I (0.04 µg/ml) and collagen IV (0.25 µg/ml). HT1080 stable cells were serum-starved for 20 h and then seeded in 96-well plates (10⁵ cells/100 µl/well) for 1 h at 37 °C. Non-adherent cells were washed away carefully with HBSS and the attached cells were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate trihydrate (pH 7.2) buffer for 20 min. Following extensive washes, attached cells were stained with 1% methylene blue in 0.01 M sodium tetraborate buffer for 30 min. Excess dye was washed off with water, and cells were lysed with 75% ethanol to be measured at 595 nm (TECAN, M€annedorf, Switzerland)²³. When necessary, cells were incubated in the presence of the GRGDSP or GRADSP peptide (50 µg/ml).

For the cell aggregation assay, serum-starved stable HT1080 cells (5 × 10⁵ cells/mL) were suspended in serum-free MEM containing 5% BSA and incubated in 96-well plates (100 µl/well) for 1 h at 37 °C. Subsequently, cells were fixed with 1% formaldehyde in PBS and counted with a hemocytometer. The equation to determine the percentage of cell aggregation was (1 − (N1/N0))×100% where N1 is the number of single cells after incubation and N0 is the number of single cells at the beginning of the experiment.