Cell apoptosis was detected using the Annexin V-phycoerythrin (PE)/7-amino-actinomycin D (7-AAD) Apoptosis Detection Kit (Nanjing KeyGen Biotech, Nanjing, China) according to the manufacturer's instructions. Cells were seed in six-well plates at 5×105 per well. The cells were harvested and washed twice in PBS. Then, 1×106 cells were resuspended in 500 μl binding buffer. The suspension was stained with 1 μl Annexin V-PE in the dark for 10 min at room temperature, and 5 μl 7-AAD was added to the suspension and maintained for 10 min at room temperature in the dark. Cell apoptosis was analyzed using a BD FACSAria II cell sorter (BD Biosciences, Franklin Lakes, NJ, USA).