Fig. 1

Detection of HIF-1α and HIF-dependent proteins in human breast tumor tissue. VEGF was measured by ELISA and HIF-1α, CA-IX and BNIP3 were analysed in tumor and matching normal breast tissue by western blotting (a). For quantification of the proteins on western blots, band density was measured with ImageJ. Levels of individual proteins were normalised to the internal loading control, β-actin. A positive control (MDA-MB-231 cells exposed to 1% O₂) was loaded onto each gel to compare between blots and used to normalise development of protein bands between gels. The data in (a) are shown to exemplify the variable expression of HIF-1 pathway proteins in three individual patients (P1-P3) in tumor (T) and normal (N) tissue. Quantification was carried out with bands below saturation levels. b, c Relative expression of HIF-1α and downstream proteins for individual tumors are shown. Grade 1 (⃞) (n = 9), Grade 2 (●) (n = 20), Grade 3 (▲) (n = 22) tumors are indicated. The HIF Pathway score (d) for each tumor sample was derived by combining the individual scores for each protein from B and C, as follows: HIF-1 pathway score = HIF-1α + CA9 + BNIP3 + VEGF. e, f, g HIF-1 dependent proteins and HIF-1 Pathway score by tumor grade, showing means ± S.D., and uninvolved tissue in comparison. Significant difference across the three grades was assessed by ANOVA