Apoptosis Assay

To determine the level of cellular apoptosis, flow cytometry analysis was performed using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ). After treatment with doxorubicin for 48 hr, cells were washed twice with prechilled PBS, and then 5 × 10⁵ cells were resuspended in 500 μL 1× binding buffer and mixed with 5 μL FITC Annexin V and 5 μL PI at room temperature (25°C) while avoiding light for 30 min. The stained cells were then counted using a fluorescence-activated cell sorting (FACS) flow cytometer (BD Biosciences, San Jose, CA).