Flow cytometry analyses

For cell cycle analysis, MDA-MB-231 and MDA-MB-468 cells were seeded in the 6-well plates with 500,000 cells/well, and then incubated at 37°C in a humidified incubator with 5% CO₂. After transfection for 72 hours, the cells were collected, washed with Dulbecco's phosphate buffered saline (DPBS; Genview, El Monte,USA), fixed in 70% ethanol, and incubated overnight at −20°C. Ethanol was removed by centrifugation. The cell pellets were washed with DPBS, followed by incubation with 100 μL propidium iodide (PI, Sigma, St.Louis, USA) solution for 5–10 minutes in the dark at 37°C. The cells were analyzed using a flow cytometer (Beckman Coulter, Brea, USA).

For apoptosis analysis, cells were stained using the Annexin V-FITC Apoptosis Detection Kit (ShanghaiGenechem Biotech Co., Ltd.) and PI staining. Cells were harvested 72 hours after transfection, binding buffer (5 μL Annexin V/FITC) was added according to the manufacturer's instructions. After 15 minutes, PI staining was performed in the dark at room temperature, followed by incubation in the dark at room temperature for another 15 minutes. Apoptosis was then detected by flow cytometry.