Cell culture and drug treatment

The U87MG, T98G, A172, U138 and LN-18 GBM cell lines were obtained from American Type Culture Collection. T98G-based model described in [56] was used, where cells were transfected with plasmid vector encoding shRNA against MGMT (T98/shRNA) or with empty vector (T98/EV). The laboratory of Dr. Thierry Muanza (McGill University) kindly provided U87MG cells stably transfected with a plasmid carrying exogenous MGMT (U87/MGMT) or an empty vector (U87/EV) (transfection by Dr. Jad Ashami at the laboratory of Dr. Rolando Del Maestro). Established GBM cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; standard medium). GBM specimens used in this study were obtained from patients undergoing surgical treatment at the Montreal Neurological Hospital, in accordance with Institutional Review Board (IRB)-approved protocols. The diagnosis of GBM was made by a neuropathologist. GSCs isolated from cancer specimens were established and grown in neurosphere cultures as previously described [58]. GSCs expanded in neurosphere cultures retained self-renewal capacity in serum-free media, expressed neural stem cell markers, such as CD133 and nestin, and had the ability to differentiate in serum-containing growth media. 48EF GSCs were kindly provided by Dr. Samuel Weiss (University of Calgary). GSCs were maintained in neural stem cell complete medium NeuroCult NS-A Basal Medium with NeuroCult NS-A proliferation supplement (STEMCELL Technologies Inc., BC, Canada), Heparin (STEMCELL Technologies, BC, Canada), Epidermal Growth factor (EGF, 20 ng/ml) and Fibroblast Growth factor 2 (FGF-2, 20 ng/ml) (Life Technologies Inc., ON, Canada). All cell lines were grown at 37°C in a humidified atmosphere containing 5% CO₂. Cells were treated with PRIMA-1^MET (Tocris Bioscience, Bristol, UK) dissolved in DMSO at varying doses in standard medium for 24 hours and then left in drug-free medium for additional time depending on the assay used. Cells treated with DMSO were used as a control.