Rhegf

U251, LN229, A172, LN18 and T98G cells were incubated in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS), purchased from the American Type Culture Collection (ATCC). Primary human N3 and K3 GBM cells were obtained as follows: Two primary human GBM samples were washed, acutely dissociated in oxygenated artificial cerebrospinal fluid and subjected to enzymatic dissociation, as described previously^{45 (link)}. HBMVECs (Sciencell) and neurospheres were incubated as described previously^{24 (link)}. NHAs were grown in the astrocyte growth media supplemented with rhEGF, insulin, L-glutamine, GA-1000, ascorbic acid and 5% FBS, purchased from Lonza. To assure the authenticity of the cell lines, we prepared frozen stocks from initial stocks and used a new frozen stock for the experiments every 3 months. To induce GSC differentiation, GSCs were dissociated and cultured on polyornithine and fibronectin double-coated plates in differentiating conditions (Neurobasal media supplemented with N2, B27, 3 mM L-glutamine, and 5% FBS)^{46 (link)}. Sources and concentrations of the reagents used were 100 nM Calp C (Merck, Whitehouse Station, NJ, USA) and 25 to 400 μM TMZ (Sigma-Aldrich, St. Louis, MO, USA).

Primary normal human astrocytes were obtained from Lonza. Cells were cultured in astrocytes medium supplemented with 0.1% rhEGF, 0.25% Insulin, 0.1% Ascorbic Acid, 0.1% GA-1000, 1% L-Glutamine, and 3% FBS (all supplements from Lonza, Germany).