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1 C Supporting Figure 1. Experimental design of s between and cells. (A) -hepatocytes were isolated from a 30 days of -progenitors. Differentiation into mature hepatocytes was achieved following a 2-weeks DMSO treatment between day 15 and day 30. () Phase contrast microphaphs of hepatocytes (left) and cells (right) after 48 hrs of. (C) -hepatocytes and cells were d alone or side by side using trawell ierts. After 48hrs, total RNA was extracted from and experiments and subjected to a microarray analysis. CM was simultaneously collected and stored at -80 C.

2 signature: 1% highly expressed genes in (n=163 genes) nb of genes 1% highly expressed genes (n=163) hits P<0.01 DL rat model Sham relative expression signature: genes related to the traformation of quiescent stellate cells into myofibroblasts (n= 97 genes) P<0.05 hits High expression in Low Supporting Figure 2. Validation of as a model of human activated hepatic stellate cells (HSC) by genomic analysis. (A) Distribution of gene expression in. cells were profiled using pangenomic microarrays and genes were ranked based on their relative expression. Genes ranked in the top 1% of highly expressed genes in all experiments (n=3) were further selected (163 genes) and submitted to Ingenuity Pathway Analysis (IPA). The top canonical pathway identified by IPA was related to hepatic fibrosis and HSC activation (P<0.0001) and the top gene network included wellknown genes involved in inflammation, ECM remodeling, HSC activation, and fibrosis (e.g. ACTG2, COL1A1, CXCL12 (SDF1), FGF2, IL1, MMP1, MMP2, PDGFR). () Gene set enrichment analysis (GSEA). Upper panel: GSEA demotrated that the 163 genes highly expressed in were significantly enriched in the hepatic gene profiles established in rats submitted to bile duct ligation (DL), a model of hepatic fibrosis [1]. Lower panel: GSEA demotrated that the genes which sign the traformation of quiescent HSC into myofibroblasts [2] were significantly expressed at a high level in cells. [1] Hepatology. (2010), 51(3): [2] J. iol. Chem. (2006), 281(24):

3 Supporting Figure 3. Cellular and extra-cellular organization of genes included in the / signature. The abundance of an mrna for a protein depicted in red or green was respectively up- or down-regulated in after 48 hrs with cells. Genes were annotated using the official gene symbols (

4 ladder Cancer (Recurrence) MYC targets Invasive reast Cancer R inactivation (fibroblasts) Advanced Gastric Cancer IL6 signaling (fibroblasts) Metastatic stromal cells Acquired therapy resistance (breast) Highly metastatic pancreatic cancer cells Silenced epigenetically (TSA) in pancreatic cancer Supporting Figure 4. Uupervised gene set enrichment analysis in gene expression profiles of and under or condition. Uupervised GSEA was done with the whole C2 collection of curated gene sets from the molecular signatures database (MSigD). Enrichment score was determined after 1,000 permutatio. Relevant gene signatures were retrieved when a significant enrichment (P<0.01) was observed both in and under the condition. Notably, in / s this approach demotrated a specific enrichment of gene signatures associated with a poor prognosis in cancer other than HCC (e.g. breast, pancreas) as well as an association with genes silenced by Trichostatin A (TSA) in pancreatic cancer.

5 P<0.001 HCC (9 months) Moderate dysplasia (3 weeks) CLUSTER 3 CLUSTER 1 Surrounding non-tumor liver (9 months) Severe dysplasia (3 months) CLUSTER 2 Supporting Figure 5. / signature correlates with HCC progression in Myc/Tgfa tragenic mice. (A) Dendrogram overview of and / experiments integrated with 80 cases of HCC derived from 4 tragenic mouse models of HCC (Myc, E2f1, Myc/E2f1 and Myc/Tgfa) [1,2]. Clustering analysis was based on the expression of genes which signed the crosstalk between and ( vs /). Two clusters (1 and 2) were identified. Cluster 2 included / samples and all Myc/Tgfa HCC. This result points out Myc/Tgfa mice as a suitable in vivo model for evaluating the relevance of / signature in HCC oet and progression. () Multidimeional scaling analysis of mouse liver tissues during the course of HCC development in Myc/Tgfa tragenic mice. Gene expression profiles were established previously using liver samples from Myc/Tgfa tragenic mice collected at various time-points ranging from 3 weeks (moderate dysplasia), 3 months (severe dysplasia) and 9 months (HCC) [2]. Here, the samples were clustered by a multidimeional scaling approach using the expression of genes included in the / crosstalk signature. Three main clusters were identified using the first 3 principal components. Cluster 1 included livers with early moderate dysplasia (cyan). Cluster 2 included livers with late severe dysplasia (dark blue) and surrounding non-tumor tissues (green). Of note, we previously demotrated that these two tumor stages exhibited similar gene profiles [2]. Cluster 3 included all fully developed HCC (red). The statistical significance test was based on a null hypothesis that the expression profiles come from the same multivariate Gaussian (normal) unimodal distribution that represents a single cluster. This results demotrate that the / signature was able to discriminate the mouse samples on the basis of HCC progression. [1] Hepatology. (2006), 44(4): [2] Carcinogenesis. (2011), 32(10):

6 Supporting Figure 6. Trichostatin A (TSA) impacts / crosstalk. (A) Expression of AREG and EREG genes in under (white bar) or (black bar) conditio in absence (-TSA) or presence (+TSA) of 500 nm TSA. Shown is relative mrna levels after 48hrs cell (mean±sd; n=3). -induced up-regulation of AREG and EREG was abolished in presence of TSA. () In vitro angiogenesis assay using HUVEC grown on a Geltrex matrix in presence of conditioned medium (CM) derived from the of (-CM) or the of with (/-CM) in absence (upper 2 panels) or presence (lower 2 panels) of 500 nm TSA. After 6hrs, tube formation by HUVEC in wells corresponding to treatment with /-CM was abolished in presence of TSA. Q-RT-PCR analysis demotrated that induction of VEGFA in under condition (black bar) was abolished in presence of TSA. ( P<0.05 ; two-tailed Student s t-test).

7 Supporting Figure 7. Evaluation of / crosstalk specificity and effect of long term conditio on IL6, IL8, VEGFA and MMP9 gene expression. (A) Comparison of IL6 (left panel) and IL8 (right panel) mrna levels detected by Q-RT-PCR in 3 hepatoma cell lines (, Huh7, HepG2) and 1 non-hepatocyte cell line (HuG, biliary cells) [1] after 48 hrs of (white bar) or with (black bar) (mean+/-sd; n=3). Up-regulation of IL6 under condition, as seen in, was not observed for the other cell lines. Surprisingly, IL6 was even found to be repressed in Huh7 d with. In contrast, the up-regulation of IL8 under condition with was observed for all hepatoma cell lines. () Evaluation of / crosstalk after 8 days of (white bar) or (black bar) (mean+/-sd; n=3). Left panel: Q-RT-PCR analysis of IL6 and IL8 in. Up-regulation of both genes under condition was maintained. Right panel: Q-RT-PCR analysis of VEFGA and MMP9 in. VEGFA up-regulation under condition was maintained after 8 days. ( P<0.05 ; two-tailed Student s t-test). [1] J. Hepatol. (1997), 26(6):

8 KEGG_HEDGEHOG_SIGNALING_PATHWAY Enrichment Score P=0.43 / IOCARTA_SHH_PATHWAY Enrichment Score P=0.41 / C Enrichment Score KEGG_HEDGEHOG_SIGNALING_PATHWAY P=0.62 / Enrichment Score IOCARTA_SHH_PATHWAY P=0.34 / Supporting Figure 8. The Hedgehog pathway in / crosstalk. (A) Expression of specific genes from the Hedgehog signaling pathway in and under (white bar) or (black bar) conditio. Shown is relative mrna levels (mean±sd; n=3). No difference was observed between and condition (P>0.05). () and (C) Uupervised analysis of the Hedgehog pathway by GSEA using the gene expression profiles of () or (C) under or conditio.

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