Research Paper Volume 12, Issue 23 pp 23478—23496

Figure 1. Increased redox status, upregulated HIF-1α and TGF-β/Smad3 pathways as well as Tim-3 and galectin-9 expression in breast tumour tissues compared to non-transformed peripheral tissues. The proposed pathway studied is summarised in panel (A), where it is indicated that xanthine oxidase (XOD) and NADPH oxidase (Nox) produce ROS which activate AP-1 transcription factor through ASK1-controlled MAP kinase cascades. HIF-1 and AP-1 contribute to the activation of TGF-β expression, which then displays autocrine activity and stimulates the activation of galectin-9 and possibly Tim-3 expression through Smad3 transcription factor. Tissue lysates were subjected to measurement of xanthine oxidase and NADPH oxidase activities as well as TBRS levels (B). HIF-1α accumulation, tissue-associated TGF-β and phospho-S423/S425-Smad3 levels (C) as well as levels of tissue-associated Tim-3 and galectin-9 (D) were analysed in tissue lysates. All quantities are expressed in respective units per 1 gram of the tissue. Normalisations against total protein loaded (for Western blot; measured by Li-Cor protein assay kit) and per mg of the total protein for enzyme activities and TBRS assays were also performed. These results are presented in the Supplementary Figure 1. Images are from one experiment representative of five which gave similar results. Data are shown as mean values ± SEM of five independent experiments. * - p < 0.05 and ** - p < 0.01 vs non-transformed peripheral tissue abbreviated as HT (healthy tissue).