Hbmvec

Primary cultures of Human Brain Microvascular Endothelial Cells (HBMVEC) were purchased from ScienCell Research Laboratories (ScienCell Research Laboratories, San Diego, CA, USA) and maintained at 37°C in a humidified atmosphere containing 5% CO₂ (v/v). HBMEC were cultured in Endothelial Cell Medium (ECM). To further assess the effects of ER stress activation and autophagy after SCI, cells were treated with TG (10 μM), 4-PBA (1 mM) and PBA compound with chloroquine (100 μM) or chloroquine alone. All experiments were performed in triplicate.

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).