4.7. DNA Content and Cell Cycle Analysis

After pevonedistat incubation (0–72 h), cells were harvested and fixed with 70% ethanol overnight. Bromodeoxyruridine (BrdU) analysis was performed using a BrdU kit (BDBiosciences, San Jose, CA, USA) according to the manufacturer’s instructions. For cell cycle analysis, cells were trypsinized, collected, and resuspended by gentle vortex while drop wise adding 5 mL of 95% ethanol. Cells were fixed for 30 min and resuspended in 1 mL of diluted PI (final PI concentration 0.5 µg/mL). PI was reconstituted with PBS from a 500 µg/mL stock solution. RNase 1 mg/mL was added and cells were incubated at 37 °C for 30 min prior to flow cytometry analysis. Data were analyzed using FlowJo (FlowJo, LLC, Ashland, OR, USA).