Flow cytometry assay

The apoptotic analysis was determined using an Annexin V/dead cell apoptosis kit (cat. no. V13241; Invitrogen; Thermo Fisher Scientific, Inc.). Samples were gently suspended in 100 μL binding buffer containing 5 μL of Annexin V-FITC and 5 μL propidium iodide (PI), and further incubated for 15 min in the dark at room temperature. Finally, cells were suspended in 500 μL binding buffer and detected by flow cytometry using FACSVerse (BD Biosciences, San Jose, CA, USA). The apoptotic rate was determined for each condition as follows: Apoptotic rate = (early apoptotic rate + late apoptotic rate) × 100%.

Cells were washed with ice-cold PBS and fixed in cold 70% ethanol overnight. Prior to analysis, cells were washed with PBS and incubated with PI for 30 min at room temperature in the dark. The DNA contents were analyzed with Multicycle for Windows using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA).