Cell apoptosis assay

Flow cytometry was performed using a FITC/Annexin V Apoptosis Detection Kit I (BD Pharmingen™) according to the manufacturer's protocols. In brief, following transfection for 48 h, cells were washed with PBS and resuspended in 100 µl annexin binding buffer (1X), followed by incubation with 5 µl FITC Annexin V and 5 µl PI working solution at room temperature for 15 min. Subsequently, 400 µl annexin binding buffer (1X) was added to the mixture. Finally, the apoptosis rate of cells was analyzed by flow cytometry using a BD FACSCalibur system (LSRFortessa X-20; BD Biosciences). Data were analyzed using FCSalyzer v16.0.153 software (Huajun Software Park).