Cell apoptosis and death assay

For cell apoptosis assay, GBM cells in serum-free medium were treated with juglone (0, 20, and 40 μM) for 48 h, 1 × 10⁵ cells were harvested and incubated in 100 μL labeling solution (5 μL of Annexin V FITC, 5 μL of PI, 10 μL of 10 × binding buffer and 80 μL of H₂O) in darkness at room temperature for 15 min, after that, 400 μL of binding buffer was added to stop the staining reaction. For cell death assay, the cells were pretreated with or without NAC (Sigma Aldrich, 2 mM), or SB203580 (Sigma Aldrich, 5 μM) for 1 h. Then the cells were treated with juglone (0, 40 μM) for 48 h. Following incubation, cells were collected and fixed in 70% ethanol for 24 h at 4 °C. After that, the cells were resuspended in 500 μL phosphate buffer solution (PBS) containing RNaseA (10 mg/mL, 50 μL) and PI (2 mg/mL, 10 μL). The mixture was incubated in the dark at 37 °C for 30 min. For cell apoptosis and death assay, cells were then analyzed on a FACS Calibur cytometer (Becton Dickinson, San Joe CA). The data were analyzed using FlowJo software V6.0 (Tree star, Ashland OR). Early apoptotic cells are defined as annexin V⁺/PI⁻, whereas late apoptotic/necrotic cells are defined as annexin V⁺/PI⁺. The extent of cell death was determined by evaluating the sub G1 fraction. The experiments were triplicated.