Apoptosis Analysis of IDH1 Mutant Cells

Cell cycle analysis was performed using Muse™ cell cycle assay kit (Millipore, Burlington, MA, USA) and Mini Flow Cytometry Muse™ Cell Analyzer (Millipore, Burlington, MA, USA), as previously described [50 (link)]. Annexin-V staining was performed using Muse^TM Annexin V and Dead Cell Kit (Millipore, Burlington, MA, USA) to measure the apoptotic cells population [50 (link)]. HA-tagged IDH1-WT or IDH1^R132H overexpressing U87MG cells (1 × 10⁵ cells/well) were seeded onto a 6-well cell culture plate and further incubated with or without TSA for 72 h. Then, cells were washed and collected into fresh tubes. Collected cell pellets were mixed and stained with 100 μL Muse™ Annexin V and Dead Cell kit reagents (Millipore, Burlington, MA, USA) for 20 min at room temperature. Apoptotic cell numbers were analyzed by using Mini Flow Cytometry Muse™ Cell Analyzer (Millipore, Burlington, MA, USA).