Apoptosis and cell cycle assays:

Apoptosis was quantified by flow cytometry using Annexin V-FITC kit from BD Biosciences. 3*10⁵ SYO1 and HS-SY-II cells were seeded in a 6-well plate and allowed to attach for 24 hours. TAK-981 was added in varying concentrations and incubated for 48 hours. After incubation, the cells were trypsinized, washed in warm PBS, and resuspended in Annexin V binding buffer. Annexin V-FITC was added and incubated at room temperature for 10 minutes. The samples were then analyzed by flow cytometry using Fortessa (BD Bioscience, USA) along with FlowJo analysis software.