Flow cytometry

For DNA content measurement, cells were washed with PBS and fixated in cold 70% ethanol overnight. After resuspension of cell pellets in PBS with 1% bovine serum albumin, samples were incubated in DNA staining solution (10 μg/ml DAPI (D9542; Sigma), 0.1 mg/ml RNase A (EN0531; Thermo Fisher) and 1% bovine serum albumin in PBS) for 20 min at room temperature, followed by flow cytometry analysis on a Navios Flow Cytometer (Beckman Coulter, Brea, CA, USA). Data were analyzed using Kaluza Flow Analysis Software (Beckman Coulter).

For apoptosis detection, cells were washed with PBS and resuspended in Annexin V Binding Buffer (556454; BD Biosciences) containing 1:100 diluted Annexin V, Alexa Fluor 555 conjugate (A35108; Thermo Fisher) and 10 μg/ml DAPI (D9542; Sigma), incubated for 20 min at room temperature in the dark before flow cytometry analysis. The flow cytometry experiments were performed independently at least three times in technical duplicate.