Glioblastoma Cell Lines Transfection

Glioblastoma U251 and LN18 cell lines were cultured at 37 °C in a humidified atmosphere with 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) containing 5 g/l glucose and supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM Gln and, respectively, 10% and 5% fetal calf serum (FCS). T98 cells were cultured in DMEM 1 g/l glucose, supplemented with penicillin, streptomycin, Gln and 10% FCS. Cells were seeded in 12-well plates and transfected with Lipofectamine 2000 transfection reagent as recommended by the manufacturer (Life Technologies, Carlsbad, CA, USA). The expression vectors for wild-type and mutated IDH1 and IDH2 were described previously.^{38 (link)} U251, LN18 and T98 stable cells lines overexpressing IDH were selected using geneticin (Life Technologies). Hydroxyglutarate was purchased from Peptech (Bedford, MA, USA) and αKG, dimethyl-αKG and oxamate from Sigma (St Louis, MO, USA). For cell death experiments, 0.5 × 10⁶ cells were plated and treated the next day with 50 μg/ml ETO (Mylan, St Priest, France), 50 ng/ml TRAIL (PreproTech, Neuilly sur-Seine, France), 60ng/ml FASL (PreproTech) and 15 μg/ml cisplatin (Mylan). γ-Irradiation was carried out in a Faxitron CP160 irradiator (Faxitron X-ray Corporation, Tucson, AZ, USA) at a dose rate of 5 Gy. The number of dead cells was evaluated by FACS after incubation of 5 min with propidium iodide (Sigma).