Cell Cycle and Apoptosis Analysis

For the cell cycle analysis, the stable EC cells were synchronized in FBS-free medium for 12 h and then recovered in fresh medium for 12 and 18 h, respectively. Afterwards, these cells were fixed with 95% ethanol at 4 °C overnight. The next day, the pellets were washed with cold PBS for three times and then stained with PI/RNase buffer (BD Pharmingen^TM) for 10 min at room temperature, followed by the analysis on the flow cytometry instrument (BD Bioscience). For the apoptosis analysis, the cells were stained with Annexin-V and PI (Invitrogen) in turn at room temperature, followed by the flow cytometry analysis according to our previous performance^{29 (link)}.