GBM neurosphere cultures were maintained in Neurocult medium (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) supplemented with epidermal growth factor (20 ng/ml) and fibroblast growth factor (10 ng/ml). Cell pools and stable subclones transfected with NOTCH2 intracellular domain (NICD2) were generated as previously described [34 (link), 42 (link)]. For treatment studies, cells were plated and allowed to grow overnight in Neurocult medium; Neurocult was then replaced the next morning with medium containing γ-secretase inhibitor 18 (GSI-18) dissolved in dimethyl sulfoxide (DMSO) at the concentrations of 0.4-50 μM. RNA and protein extractions and all cell-based assays were performed 2-5 days after drug application. Cell mass was measured using CellTiter assays according to the instructions of the manufacturer (Promega, Madison, WI, http://www.promega.com). Cell number and viability were assessed using the Guava PCA and Viacount reagent according to instructions (Guava Technologies, Hayward, CA, http://www.guavatechnologies.com). All experiments were performed in triplicate.