2.2. Cytotoxicity, Cell Cycle, and Apoptosis Assay

To test cell viability after drug treatment, cells were plated at 10⁴ cells/ml in 96-well plates and treated with Cyclophosphamide and/or MLN8237 for 72h. Cell viability was assessed by Trypan Blue staining. For cell cycle assays, 10⁶-10⁷ cells were collected by centrifugation, washed twice in cold PBS, and stained with Fixable Viability Dye eFluor780 (eBioscience, San Diego, CA, USA). Cells were washed, resuspended in PBS and fixed with 0.5-1ml ethanol. Cells were reconstituted by resuspending in 1ml PBS, and samples were run on Miltenyi MACSQuant VYB (Bergisch Gladbach, Germany) and analysis done using FlowJo software (Ashland, OR, USA). For apoptosis, cells were collected by centrifugation and stained for AnnexinV-FITC and PI using Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Life Technologies; Carlsbad, CA, USA) according to manufacturer’s instructions.