Cell viability was investigated using Cell Counting Kit-8 and colony formation assays. Cell migration and invasion assays were performed using Transwell cell migration assay kits and BioCoat™ Matrigel® Invasion Chambers (Corning Incorporated), respectively. For the migration assay, 5×104 cells were seeded into the upper chamber and incubated for 24 h at 37°C in 5% CO2. For the invasion assay, 1×105 cells were plated on chambers pre-coated with 1.6 mg Matrigel (Corning, Inc.) and incubated for 36 h at 37°C in 5% CO2. Subsequently, cells were fixed, stained with 0.5% crystal violet solution at room temperature for 10 min, photographed and counted under a light microscope (magnification, ×200).
Cell apoptosis was analyzed with the Annexin V-fluorescein isothiocyanate/propidium iodide Apoptosis Detection kit. Briefly, 1×105 cells were re-suspended in 500 µl binding buffer and stained with 5 µl Annexin V and 5 µl PI in the dark at room temperature for 20 min. The samples were then analyzed using a FACScan flow cytometer and the CellQuest™ Pro 1.0 software (BD Biosciences), according to the manufacturer's instructions.
Cell apoptosis was analyzed with the Annexin V-fluorescein isothiocyanate/propidium iodide Apoptosis Detection kit. Briefly, 1×105 cells were re-suspended in 500 µl binding buffer and stained with 5 µl Annexin V and 5 µl PI in the dark at room temperature for 20 min. The samples were then analyzed using a FACScan flow cytometer and the CellQuest™ Pro 1.0 software (BD Biosciences), according to the manufacturer's instructions.