Animals were killed after tumour size had reached 10mm in diameter. Cell cultures The colon carcinoma cell lines LS174T, HT29, DLD-1 and SW948 were cultured in VLE RPMI 1640 (Biochrom, www.selleckchem.com/products/17-DMAG,Hydrochloride-Salt.html Berlin, Germany) supplemented with 10% FCS. Human umbilical vein endothelial cells were purchased from Promocell (Heidelberg, Germany) and cultured in endothelial cell growth medium (Promocell). All cell lines were maintained at 37��C and 5% CO2, except for one experiment involving exposure to controlled hypoxia (1% O2, 5% CO2, balanced N2) for 24h. Quantitative real-time PCR The RNA was extracted from tissue microdissections using the NucleoSpin RNA isolation kit (Macherey-Nagel, D��ren, Germany) and reverse transcribed using the RevertAid cDNA Synthesis Kit (Fermentas, St Leon-Rot, Germany).

For gene expression analysis in xenografts, tumours were suspended in RNAlater (Qiagen, Hilden, Germany). The RNA was extracted as described above. Real-time PCR for Ang-2 was carried out using the LightCycler FastStart DNA Master Plus Mix (Roche, Basel, Switzerland). Results were normalised to a dilution series of pre-quantified pooled PCR products with the RelQuant Software (Roche). Primer sequences for human- and murine-specific amplification of Ang-2 and ��-actin were as published (Thijssen et al, 2004). Universal GAPDH primers were as follows: Forward 5��-TGC(A/C)TCCTGCACCACCAACT-3��, Reverse 5��(C/T)GCCTGCTTCACCACCTTC-3��. Western blot For western blot (WB) analysis, cytosolic extracts of cultured cells were prepared as described previously (Kashkar et al, 2002).

Equal amounts (100��g) of protein were separated by SDS-PAGE, transferred to a nitrocellulose membrane (Schleicher & Anacetrapib Schuell, Dassel, Germany), and probed with Ang-2 antibody (F18, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Primary antibodies were detected using a horseradish-peroxidase-conjugated secondary antibody (1:2000; Dako, Hamburg, Germany) and visualised with the ECL system (Amersham Biosciences, Hamburg, Germany). Culture supernatants were analysed for Ang-2 protein concentrations using Quantikine Immunoassays (R&D Systems, Wiesbaden, Germany). Clinical samples A total of 90 patients with colorectal adenocarcinoma and 33 healthy volunteers were studied between September 2005 and November 2008. One cohort of 56 patients had newly diagnosed CRC of various stages (UICC I�CIV). After obtaining informed consent, serum samples and tumour tissues were collected at the time of primary resection (sampling from September 2005 to August 2006).