FACS (fluorescence-activated cell sorting) analysis

Cells were treated with MLN4924 for 24–72 h, harvested and fixed in 70% ethanol overnight. Cells were washed twice with ice-cold phosphate buffered saline (PBS) and then stained with propidiumiodide (PI, 20 mg/ml, Sigma) solution for 30 min in the dark. The samples were then analyzed using a BD FACScan flow cytometer facility for cell cycle distributions.

Apoptosis were detected by translocation of phosphatidylserine to the cell surface using an annexin VFITC apoptosis detection kit I (BD, Becton, Dickinson and Company, US). Cells were exposed to different concentration of MLN4924, then collected and stained with FITC Annexin V and PI according to manufacturer’s instructions prior to analysis by flow cytometry.