Cell Cycle and Apoptosis Analysis

To assess cell cycle status [39 (link)], MDA-MB-231 and MCF7 cells were seeded (1 × 10⁶ cells/well) in 6-well plates. After 48 h, cells were collected and centrifuged at 800 rpm for 5 min, then washed with phosphate-buffered saline (PBS) twice, and fixed in ice-cold 70% ethanol for at least 24 h, and treated with 100 μL of 50 mg/L propidium iodide for 30 min at 4°C in the dark. The cell cycle data were analyzed by CELL Quest software (BD Biosciences, San Jose, CA, USA). For apoptosis analyses, acridine orange/ethidium bromide (AO/EB) fluorescence staining was used [39 (link)]. Cells were re-plated in 6-well plates. After 24 h, cells were washed in PBS three times then stained with AO/EB for 5 min and visualized immediately under a fluorescence microscope (LEICA CTR4000B; Leica Microsystems, Buffalo Grove, IL, USA). The percentage of apoptotic cells was then calculated by the formula: percentage of apoptotic cell (%) = (amount of apoptotic cells/total cells examined) × 100%.