Flat bottomed tissue culture plate

To investigate the effect of PE on the migration of glioma cells, we undertook a scratch assay. First, glioma cells (A172, T98G, U87, dKGS01, and dKGS07) were seeded into a 24‐well flat‐bottomed tissue culture plate (Falcon) at a density of 1 × 10⁵ cells per 500 μL. All cells were treated with PE (5 μM), and DMSO was used as a control. After incubating them for 24 h, we checked whether all culture plates were confluent and scratched the center of all plates with a 200 μL pipette to create a cell‐free zone (wound). Thereafter, all culture plates were washed twice using PBS and the cells were treated with PE (5 μM) with DMSO as a control. Finally, 12 h after scratching the culture plates, we measured the distance of the wounds. The average wound distance was calculated from the five micrographs in each group and plotted graphically.

Apoptosis in glioma cell lines was assessed using fluorescent immunostaining. First, glioma cells (A172, T98G, U87, dKGS01, and dKGS07) were seeded into a 6‐well flat‐bottomed tissue culture plate (Falcon) at a density of 1 × 10⁵ cells per 1000 μL. Each cell line was treated with PE (5 μM) or DMSO, used as a control. After incubating them for 48 h, all culture plates were washed twice using PBS. Cells were stained with Hoechst 33,258 (1:500 dilution; Dojindo) and PI (1:500 dilution; Dojindo) for 30 min in room air. The average number of cells undergoing apoptosis from five HPFs in each group was calculated and plotted. To investigate the involvement of caspase 9 in apoptosis induced by PE, we carried out a cell apoptosis assay with pretreatment of irreversible caspase 9 inhibitor, Z‐LEHD‐FMK (MBL), as previously described.²⁸, ²⁹, ³⁰ Z‐LEHD‐FMK was used at a final concentration of 20 μM and was added 2 h prior to PE (5 μM) treatment.