The slides were then washed in PBS and mounted. Orthotopic U87ΔEGFR xenograft mouse models treated with bevacizumab or the combination of bevacizumab and cilengitide were killed at 18 days after tumor implantation (n = 3 per treatment). Approximately 40 mg of brain tumor samples were excised cleanly from each mouse, and RNA was extracted using TRIzol (Life Technologies, Carlsbad, CA) and an RNeasy Mini Kit (Qiagen, Venlo, Netherlands). They were analyzed

using a CodeLink Human Whole Genome Bioarray (Applied Microarrays, Inc, Tempe, AZ). We entrusted the microarray analyses to Filgen, Inc (Nagoya, Japan). Briefly, for each bioarray, 10 μg of biotin-labeled aRNA, which was prepared using a MessageAmp II-Biotin Enhanced Kit in a total volume of 25 μl, was added FK228 chemical structure to 5 μl of 5 × fragmentation buffer, which was then incubated at 94°C for 20 minutes. Thereafter, Ruxolitinib cost 10 μg of fragmented cRNA, 78 μl of hybridization buffer component A, and 130 μl of hybridization buffer component B were added, and the final volume was brought up to 260 μl with water. The resultant hybridization reaction mixture was incubated at

90°C for 5 minutes, after which 250 μl were slowly injected into the input port of each array, and the ports were sealed with sealing strips. The bioarrays were incubated for 18 hours at 37°C while shaking at 300 rpm. A consistent hybridization time was maintained for comparative experiments. Following the incubation, the bioarrays were washed with 0.75 Tris-NaCl-Tween (TNT) buffer Mannose-binding protein-associated serine protease (0.10 M Tris-HCl, pH 7.6, 0.15 M NaCl, 0.05% Tween 20) and incubated at 46°C for 1 hour. Each slot of the small reagent reservoir was then filled with 3.4 ml of Cy5-streptavidin working solution, and the array was incubated at 25°C for 30 minutes. Thereafter, the bioarrays were washed four times for 5 minutes each with 1 × TNT buffer at 25°C, rinsed twice in 0.1 × SSC

(Ambion, Austin, TX)/0.05% Tween 20 for 30 seconds each, and immediately dried by centrifugation for 3 minutes at 25°C. Finally, the arrays were scanned using a GenePix4000B Array Scanner (Molecular Devices, Sunnyvale, CA). A gene was defined as being upregulated when the combination therapy/bevacizumab monotherapy average intensity ratio was > 2.0, and downregulated when the combination therapy/bevacizumab monotherapy ratio was < 0.5. We performed pathway analysis on the genes with increased and decreased expression using Microarray Data Analysis Tool Ver3.2 (Filgen, Inc). The data were extracted using the following criteria: Z score > 0 and P value < .05. Total RNA was isolated from cultured U87ΔEGFR cells treated with cilengitide (1.0 μM for 16 hours) and untreated control U87ΔEGFR cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany).