RNA was reverse transcribed with oligo(dT) primers using the SuperScript III First-Strand Synthesis System for reverse transcription–polymerase chain reaction (RT-PCR; Invitrogen) according to manufacturer’s instructions. Primers specific to each gene were designed using the Primer3Plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) and synthesized by Invitrogen. The resulting cDNA was amplified by PCR using the gene-specific

primers and the 7300 Real Time PCR System (Applied Biosystems, Foster City, CA) and a QuantiTectTM SYBR Green PCR Kit (Qiagen, Hilden, Germany). A log-linear relationship between the amplification curve and quantity of cDNA in the range of 1 to 1000 copies was observed. The cycle number at the threshold was used as the threshold cycle (Ct). Changes in the expression of mRNA were detected from 2− ΔΔCt using the 7300 Real Time PCR System with Sequence Detection Software (version Crenolanib concentration 1.4; Applied Biosystems). The amount of cDNA in each sample was normalized to the crossing point of the housekeeping gene glyceraldehyde-3-phosphate

dehydrogenase (GAPDH). The following thermal cycling parameters were used: denaturation at 95°C for 10 minutes followed by 45 cycles at 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The relative up-regulation of mRNA for each gene in the control was calculated using their respective crossing points with GDC-0199 solubility dmso the following formula, as previously described [14]: F=2(TH−TG)−(OH−OG)F=2TH−TG−OH−OGwhere

F is the fold difference, T is control, O is the treated cell or tumor, H is the housekeeping gene (GAPDH), not and G is the gene of interest. C-src tyrosine kinase primers: CSK F (forward), 5′-GAATACCTGGAGGGCAACAA-3 Caveolin 3 primers: CAV3 F (forward), 5′-TTTGCCAAGAGGCAGCTACT-3 GAPDH primers: GAPDH F (forward), 5′- GAGTCAACGGATTTGGTCGT-3 To assess the gene expression of caveolin 3 and c-src tyrosine kinase with quantitative RT-PCR, athymic mice harboring U87ΔEGFR brain tumors were killed at 18 days after tumor implantation. The tumor-bearing right hemispheres of the brains were excised and processed for RNA. Student’s t-test was used to test for statistical significance. Data are presented as the means ± standard error. P < .05 was considered statistically significant. All statistical analyses were performed with the use of SPSS statistical software (version 20; SPSS, Inc, Chicago, IL). U87ΔEGFR orthotopic tumors proliferate with aggressive angiogenic growth (Figure 1A). Treatment with bevacizumab reduced angiogenesis in U87ΔEGFR orthotopic tumor tissues in the rat with a regression of tumor size ( Figure 1B). The density of tumor vessels was significantly decreased by bevacizumab ( Figure 1C). The short diameter of tumor vessels tended to be larger, but there was no significant difference ( Figure 1D).