Human microvascular endothelial cells (HMEC-1), kind gift from Centre for Disease Control and Prevention, Atlanta, U.S.A., were grown in MCDB 131-medium (Gibco) supplemented with 5% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Gibco), 10 ng/ml epidermal growth factor (ICN, Costa Mesa, CA, U.S.A.) and 1 μg/ml hydrocortisone (ICN), and maintained on uncoated dishes in a 5% CO
2/95% air atmosphere in a humidified incubator at 37°C. Porcine aortic endothelial cells stably transfected with KDR (PAE-KDR), provided by Shay Soker, Winston-Salem, NC, were maintained in F-12/HAM medium supplemented with 5% fetal bovine serum at 37°C in 5% CO
2/95% air. Commercial human umbilical vein endothelial cells (HUVECs) (Lonza) were cultured in EGM-2 medium (Clonetics) including 2% fetal calf serum. The human glioblastoma cell lines G28 and G44 [
24,
25], kindly provided from the Department of Neurosurgery, University Hospital Hamburg-Eppendorf, were cultured in Modified Eagle's Medium supplemented with 10% fetal bovine serum on uncoated dishes. Cilengitide (CGT) was kindly provided by Merck Serono, Darmstadt, Germany. Stock solutions were diluted in sterile physiological saline solution at 20 mg/ml. Cells were incubated with cilengitide in final concentrations of 1, 5 and 50 μg/ml. Temozolomide (TMZ) was purchased from Bristol Myers Squibb, Munich. Stock solution was diluted in DMSO at 5 mg/ml. Cells were treated with temozolomide in a final concentration of 5 μg/ml. Texas Red-X phalloidin was from Invitrogen, mouse monoclonal anti phospho-Akt (Ser473) antibody was from Cell Signaling, rabbit polyclonal anti phospho-Src (Y418) antibody was from Biosource, mouse monoclonal anti phospho-Src (Y416) was from Biomol, mouse monoclonal anti Src antibody was from Upstate (NY, USA), mouse monoclonal anti phospho-FAK (Y397) was from BD Biosciences, rabbit polyclonal anti ZO-1, anti Erk1/2, mouse monoclonal anti phospho-Erk and anti β-actin antibodies were from Santa Cruz Biotechnology.
Tissue culture plates were incubated with a 12 mg/ml polyHEMA (Poly(2-hydroxyethyl methacrylate); Sigma Aldrich) ethanol solution at 37°C or with 10 μg/ml fibronectin (Harbor Bio-Products, Norwood, MA, USA) at 4°C overnight when indicated.