U251 cell line culture

U251 cells ware cultured at 37°C in 5% CO₂ in high glucose DMEM and 10% fetal bovine serum supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml). In order to evaluate proteasome involvement in corilagin-induced cell differentiation, cells were treated with proteasome inhibitors MG-132 (0, 200 nM, 1, 5 µM) and bortezomib (0, 5, 40, 100 nM) for 72 h and carfizomib (0, 100, 200 nM, 1 µM) and PR-957 (0, 100 nM, 1, 10 µM) for 24 h at 37°C. In order to prove whether corilagin increases TMZ-induced apoptosis of U251 cells, flow cytometry was performed using U251 cells treated with increasing concentrations of corilagin (0, 25, 50, 100 and 200 µg/ml) and/or TMZ (0, 25, 50, 100 and 200 µg/ml) for 72 h at 37°C. In order to demonstrate that proteasome inhibition mediated corilagin-induced apoptosis, flow cytometry was performed using U251 cells treated with corilagin + MG-132 (200 nM) and corilagin + bortezomib (5 nM) for 72 h at 37°C.