Cell cycle and apoptosis assay

Cells were cultured with three gradient densities of ALO in a six-well plate for 48 h. For the cell cycle assay, cells were collected and fixed with 75% cold ethanol at −20°C overnight. Subsequently, cells were stained with 50 μg/mL of propidium iodide (PI) and 1 mg/mL RNase for 30 min at 4°C. Cell cycle distributions were analyzed using a FACS flow cytometer (BD Biosciences, San Jose, CA, USA). As for the apoptosis assay, cells were stained with Annexin V-FITC/PI which was contained in an apoptosis kit (KeyGene Biotech). Flow cytometry was then performed and results were analyzed.