Examination of protein expression For the HIF-1α ELISA, cells were harvested and lysed in 50 mM TRIS, 300 mM NaCl, 3 mM EDTA, one mM MgCl2, 25 mM NaF, 20 mM β-glycerophosphate, 1% Triton-X, 10% glycerol and protease inhibitor cocktail P-8340 Sigma, St Louis, MO, USA. Total protein was quantified by the Bicinchoninic assay BCA Pierce, Rockford, Inhibitors,Modulators,Libraries USA. The HIF-1α Duoset IC R&D Systems, Minneapolis, USA was used to measure HIF-1α protein in complete protein ly- sates. Results were analysed using Ascent Version 2.6 soft- ware Thermo Fisher Scientific, Waltham, MA, USA. Western blotting was performed using total protein lysates from cells harvested and lysed with urea buffer 8 M urea, 1% Sodium Dodecyl Sulphate, 1% glycerol and 10 mM Tris pH6.8 0.

5 mM protease inhibitor cocktail Sigma-Aldrich, Poole, UK 1 mM dithiothreitol for HIFs, or RIPA buffer 50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X, 0.1% SDS, 5 mM MgCl2, 50 mM NaF, 50 mM DTT, 2 mM orthovanadate, 5 mg mL sodium deoxycholate, ten mM sodium pyrophosphate, ezh2 protein inhibitor 25 mM β-glycerophosphate, 2 mM EDTA, 2 mM PMSF and pro- tease inhibitor cocktail P-8340 Sigma, St Louis, MO, USA for signalling studies. Samples have been resolved on SDS-polyacrylamide gels, where a 3-8% Tris-Acetate NuPAGE? Novex gel Invitrogen, Carlsbad, CA, USA was used for EGFR signalling studies, and a 4-12% Bis-Tris NuPAGE? Novex gel Invitrogen, Carlsbad, CA, USA was used for signalling and HIF-α protein studies. Rabbit anti-human phospho EGFR Tyr 1068 phospho EGFR Tyr 845 phospho p38 MAP Kinase Thr 180 Tyr 182 phospho p44 42 MAP Kinase Thr 202 Tyr 204 phospho-Akt Ser 473 complete EGFR, complete p38 MAPK and complete p44 42 MAPK have been from Cell Signaling Technology Danvers, MA, USA.

Mouse anti-human HIF-1α and HIF-2α EPAS had been from Becton Dickinson Franklin Lakes, NJ, USA and Santa Cruz Biotechnology Santa Cruz, CA, USA respectively. Secondary anti-rabbit and mouse HRP-conjugated antibodies have been from Dako- Cytomation Glostrup, Denmark. Whole cell lysate of EGF-treated Sunitinib solubility A431 epithelial carcinoma cells used as posi- tive control was from Santa Cruz Biotechnology Santa Cruz, CA, USA. Densitometry was performed using Phoretix 1D analysis software TotalLab Ltd, Newcastle upon Tyne, UK. Statistical analyses Statistical significance was evaluated with 1-way ANOVA with Dunnett’s post-hoc test to compare selected groups of data.

The ΔCt values had been used to determine the sta- tistical significance of differences between groups for PCR-based studies. 2-way ANOVA with Bonferroni cor- rection was used to compare selected groups of data with respect to time. Results HIF-dependent induction of angiogenic genes in Caco-2 cells in response to hypoxia and the hypoxia mimetic DMOG Since hypoxia is likely to be a key stimulus for angioge- nesis in CRC, we first investigated the angiogenic gene profile of Caco-2 cells exposed to either hypoxia or the hypoxia mimetic DMOG. Figure one and Table one illustrate the Human Angiogenesis RT2 Profiler? PCR array data as scatter plots, and show that 9 pro-angiogenic genes have been significantly changed by a factor of at least 2.0-fold in response to either hypoxia Figure 1a or DMOG Figure 1b including VEGF-A, known to be highly regu- lated by hypoxia in various cell types fold increase three.one and 3.4 in response to hypoxia and DMOG respectively.