Flow cytometry analysis of cell cycle

The transfected HA-VSMCs were cultured in 6-well plates. To perform cell cycle analysis, the cells were digested with trypsin and fixed with 70% ethanol for 24 h at 4°C. The cells were stained according to the manufacturer’s protocol of CycleTEST PLUS DNA Reagent kit (BD Biosciences). The fixed cells were washed 3 times with PBS and centrifuged at 300 × g for 5 min in room temperature. The supernatant was subsequently discarded. Cells were incubated with propidium iodide (PI) stain buffer for 10 min and subsequently treated with 200 µl of PBS, 200 µl of RNase A and 200 µl of PI for 10 min at 4°C in the dark. Finally, the cells were screened by 400-mesh sieves and analyzed by flow cytometry (FACSCanto II; BD Biosciences). The results obtained were analyzed using the ModFit software (version 4.1; Verity Software House, Inc., Topsham, ME, USA).