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Supplementary material for this article can be found on the H EPATOLOGY Web site ( http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html ).

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hep22283-SupplementaryFigure1.tif69K Supplementary Fig. 1.Hepatocytes isolated fromTgfbr2KO livers are insensitive to the growth inhibitory effects of TGF-β. Cell viability of hepatocytes isolated from WT and KO livers was estimated after 24, 48, 72 and 96 hours of treatment with 1 ngmL of TGF-β. TGF-β treatment resulted in a more than 60% reduction in viability of control hepatocytes. Escape from the growth inhibitory effect of TGF-β in KO hepatocytes confirms the deregulation of TGF-β signaling in hepatocytes deficient for theTgfbr2gene. Cell viability is expressed as a percentage over the control. Columns, mean; bars, SEM, n=3.
hep22283-SupplementaryFigure2.tif3247K Supplementary Fig. 2.Genes persistently differentially expressed between WT and KO hepatocytes regardless of TGF-β treatment. In the absence of TGF-β treatment (two left panels), 417 genes showed a significant and persistent expression difference between WT and KO hepatocytes (P 0.01, random-variance t-test, 1.5 fold difference). Genes clustered in two main groups as their expression was lower (Cluster 1) or higher (Cluster 2) in KO hepatocytes compared to the WT hepatocytes. These genes were not included as part of the TGF-β signature because their induction or repression were not dependent on TGF-β treatment (as judged by the similarity of the two left vs. two right panels). The differential expression of these genes may reflect a long-term adaptation of KO hepatocytes to the loss of a functionalTgfbr2receptor.
hep22283-SupplementaryFigure3.tif3088K Supplementary Fig. 3.Ingenuity Pathway Analysis identifies TGF-β as the central regulator of genes included in the mouse signature. Upper part List of genes included in the top-rank canonical pathway identified by the Ingenuity Pathway Analysis tool (TGF-β signaling, score 41, P = 4.75.10 -6 ). The score is determined by the number of differentially expressed genes within each networks and the strength of the associations among networks. Lower part Graphical view of genes interconnected with TGF-β. Genes up- and down-regulated by TGF-β are respectively colored in red and green.
hep22283-SupplementaryFigure4.tif3695K Supplementary Fig. 4.Mouse TGF-β signatures identify distinct tumor subtypes in an independent set of human HCC. (A) Dendrogram and heat-map overview of mouse TGF-β signatures integrated with an independent dataset including 104 human HCC and 7 liver metastasis. Clustering is based on the expression of 176 orthologous genes, and the data are presented in a matrix format in which rows and columns represent respectively genes and samples. (B) Detailed view of mouse and human samples cluster analysis. By using the same criteria as in Fig. 2, integration of mouse and human datasets identified three subtypes of HCC characterized by late (red), early (blue) and negative (pink) TGF-β signatures. All liver metastasis clusters with the late TGF-β signature.
hep22283-SupplementaryFigure5.tif190K Supplementary Fig. 5.HCC-derived cell lines harboring a late TGF-β signature overexpress genes involved in TGF-β induced cell motility, metastasis and EMT. Gene expression patterns of selected genes were compared in cell lines harboring early vs. late TGF-β signatures. Gene expression is expressed as a percentage over the cell lines harboring an early TGF-β signature (Columns, mean; bars, SEM). TGF 1, transforming growth factor, beta 1; SMAD2, SMAD family member 2; TGIF2, TGF-β -induced factor homeobox 2; SNAI1, snail homolog 1; TWIST1, twist homolog 1; VIM, vimentin; MMP1, matrix metallopeptidase 1; CD44, CD44 molecule; MTSS1, metastasis suppressor 1.
hep22283-SupplementaryFigure6.tif2928K Supplementary Fig. 6.Early and late TGF-β signatures predict prognosis in both liver and lung cancer. (A) Dendrogram overview of early and late mouse TGF-β signatures integrated with the gene expression profiles of human HCC and lung adenocarcinomas. Clustering is based on the expression of 174 orthologous genes. Tumors which cluster with mouse samples corresponding to WT hepatocytes challenged with TGF-β for 0.5-2h (early signature) or for 4-24h (late signature) are respectively colored in blue and red. (B) Clinical relevance of tumor subtypes defined by the TGF-β signatures. Kaplan-Meier plots and log-rank statistics demonstrate the difference in the overall survival between individuals with tumors harboring early (blue) or a late (red) TGF-β signatures.
hep22283-SupplementaryFigure7.tif1643K Supplementary Fig. 7.Expression of genes associated to cell-cycle, metastasis and angiogenesis in human HCC harboring early and late TGF-β signatures.Functional analysis of genes differentially expressed between human HCC harboring early vs. late TGF-β signatures revealed a higher expression of genes characteristic of a high proliferation status and an invasive phenotype in HCC associated with the late TGF-β signature.
hep22283-SupplementaryFigure8.tif1594K Supplementary Fig. 8.Expression of genes associated to liver specific metabolism in human HCC harboring early and late TGF-β signatures. Functional analysis of genes differentially expressed between human HCC harboring early vs. late TGF-β signatures revealed a higher expression of genes involved in liver metabolism in HCC associated with the early TGF-β signature.
hep22283-SupplementaryFigure9.tif1694K Supplementary Fig. 9.Expression of detoxication associated genes in human HCC harboring early and late TGF-β signatures. Functional analysis of genes differentially expressed between human HCC harboring early vs. late TGF-β signatures revealed a higher expression of genes involved in detoxication and anti-oxidant defense in HCC associated with the early TGF-β signature.
hep22283-SupplementaryTable1.doc976KSupporting Information file hep22283-SupplementaryTable1.doc
hep22283-SupplementaryTable2.doc584KSupporting Information file hep22283-SupplementaryTable2.doc
hep22283-SupplementaryTable3.doc27KSupporting Information file hep22283-SupplementaryTable3.doc
hep22283-SupplementaryTable4.doc31KSupporting Information file hep22283-SupplementaryTable4.doc
hep22283-SupplementaryTable5.doc103KSupporting Information file hep22283-SupplementaryTable5.doc

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