Propidium iodide staining solution

The transfected cells were collected, washed carefully with phosphate-buffered saline (PBS), fixed with 75% ethanol, stored at − 20 °C overnight, incubated with RNAse and stained with propidium iodide staining solution (MultiSciences, Hangzhou, China) at RT for 15 min for the cell cycle analysis.
The apoptotic assay was conducted using an Annexin V-APC/PI Apoptosis Detection Kit (Multisciences, Hangzhou, China) and analyzed with a flow cytometry (FACScan, BD Biosciences). The ratio of early and terminal apoptotic cells was detected to calculate the apoptotic rate.

Apoptosis was measured by the flow cytometric detection (FACScan, BD Biosciences) of phosphatidylserine externalization using Annexin V‐APC/PI Apoptosis Detection Kit (Multiscience, Hangzhou, China). Cells were discriminated into viable cells, dead cells, early apoptotic cells, and late apoptotic cells, and then, the relative ratio of early and late apoptotic cells accounting for total cells was compared from each treated group.
Cells for cell cycle analysis were stained with propidium iodide staining solution (Multisciences, Hangzhou, China) following the protocol and analyzed by the flow cytometer mentioned above. The percentage of the cells in G0‐G1, S, and G2‐M phases were counted and compared.