Supplemental Materials

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1 Supplemental Materials Materials and Methods Histopathologic and immunohistochemical evaluation Liver tissue was divided and fixed in phosphate-buffered neutral formalin, embedded in paraffin, and cut into 5- m-thick sections. The tissues for cryostat sections were freshly collected, snap-frozen in liquid nitrogen, OCT-embedded and cut into 8- m-thick sections. The following primary antibodies were used: mouse anti- -Catenin (Santa Cruz Biotech), rat anti-a6 (a kind gift from Dr Valentina Factor, National Cancer Institute, Bethesda, MD), mouse anti-ov6 (R&D). Polyclonal antibodies reactive with -Catenin were used as described(1). Vector ABC kit (Vector Laboratories) and DAB reagent (Dako Comp) were employed in the detection procedure. For immunofluorescent staining, tissues were visualized with fluorescein-conjugated secondary antibodies (Invitrogen). All the slides were observed and photographed with an Olympus microscope (IX-70 OLYMPUS, Tokyo, Japan). In some studies, expression was quantified using Image-Pro Express software (version 5.0; Media Cybernetics, Inc.). Western Blot Assay Whole liver tissue or cultured cells were homogenized in Triton lysis buffer (20mM Tris, ph 7.4, 137mM NaCl, 10% glycerol, 1% Triton X-100, 2mM EDTA, 1mM PMSF, 10mM NaF, 5 mg/ml aprotinin, 20 mm leupeptin, and 1mM sodium orthovanadate) and centrifuged at 12, 000 g for 15 min(2). Protein concentrations were measured using the BCA assay. Immunoblotting was performed using 1:1000 anti- -Catenin (sc-7963, Santa Cruz Biotech.), 1:200 anti-c-myc (Clone 67P05, Neomarkers), 1:500 anti-cyclin D1 (Santa Cruz Biotech), 1:1000 anti- -Actin (Cell Signaling Technology) and 1:5000 anti-gapdh (Chemicon) antibodies. Immunocomplexes were detected with horseradish-peroxidase-conjugated secondary antibodies. Membranes were developed by chemoluminescence (ECL; Amersham Bioscience). RT-PCR Total RNA was prepared from liver tissue samples or cultured cells using Trizol (Invitrogen) according to the manufacturer s protocol. Reverse transcriptase PCR was performed using Superscript III RT (Invitrogen) in the presence of random primers. Amplification of the generated cdna was carried out in QuantiTect SYBR Green PCR Master Mix with ABI PRISM 7300HT Sequence 1

2 Detection System. Primer sequences are listed in the Supplemental Table 1 and 2 (Supplemental Data). Each measurement was performed in triplicate and the results were normalized to the expression of the -actin (rat) or 18S (human) reference gene. Luciferase assay Two different Tcf-luciferase reporter constructs were used in this study: an intact wild-type Tcf-luciferase construct (pgl3-ot) and a mutant Tcf-luciferase reporter construct (pgl3-of) (both gifts of B. Vogelstein) (3). The pgl3-ot promoter comprises three LEF-1/TCF binding sites, whereas pgl3-of contains mutated inactive TCF sites and is a negative control. A Dual Luciferase reporter assay was carried out according to the manufacturer s suggestions (Promega). prl-tk (Promega) was cotransfected with each reporter construct to normalize for transfection. Luciferase activity was determined 48h after transfection. Relative luciferase activities were calculated as the OT to OF ratio. The histograms are presented as the mean ±SD from at least three independent experiments. Chromatin immunoprecipitation (ChIP) ChIPs were performed as described previously(2) with minor modification. Protein-DNA cross-linking was performed by incubating suspensions of liver with formaldehyde at a final concentration of 0.4% for 10 min at room temperature with gentle agitation. For ChIP assay, chromatin was isolated from formaldehyde-treated liver, fragmented to a mean size <600 bp and subjected to ChIP using Chromatin Immunoprecipitation Assay kits (Upstate Biotechnology, Hamburg, Germany) together with 10µg of -catenin-specific antibody following Upstate s protocol. Specific primer pair(2) for c-myc promoter region was used for investigating binding of -catenin to DNA. For analyzing chromatin input, one-fiftieth and for all other reactions one-tenth of the precipitated chromatin was taken as a template. Lentiviral vector construction and virus production -catenin expression was down-regulated by short interfering RNA duplexes. Target regions in -catenin were identified by BLOCK-iT RNAi Designer Program (Invitrogen). The mirna sequence targeting -catenin was: 5 -TGCTGTCTGCATGCCCTCATCTAATGGTTTTGGCCACTGACTGACCATTAGATGG GCATGCAGA-3,5 -CCTGTCTGCATGCCCATCTAATGGTCAGTCAGTGGCCAAAACC 2

3 ATTAGATGAGGGCATGCAGAC-3. Synthetic duplexes were cloned into the pcdna6.2gw/emgfp vector in the middle of the micro RNA 155 (mir155) sequence supplied as a part of the BLOCK-iT Lentiviral Pol II mir RNAi expression system kit (Invitrogen) to creat mirna- -catenin. The expression cassette was transferred to the lentiviral shuttle vector plenti6/v5-dest vector (Invitrogen) by Gateway recombination following the kit instructions. The VSV-G pseudotyped lentiviral vectors LV-control and the derived LV-mi- -catenin were produced by transient cotransfection of plasmid DNA and packaging plasmids into 293T cells using approved protocols. Reference List 1 Yan,H.X., He,Y.Q., Dong,H., Zhang,P., Zeng,J.Z., Cao,H.F., Wu,M.C. and Wang,H.Y. Physical and functional interaction between receptor-like protein tyrosine phosphatase PCP-2 and beta-catenin, Biochemistry, 41: , Yan,H.X., Yang,W., Zhang,R., Chen,L., Tang,L., Zhai,B., Liu,S.Q., Cao,H.F., Man,X.B., Wu,H.P., Wu,M.C. and Wang,H.Y. Protein-tyrosine phosphatase PCP-2 inhibits beta-catenin signaling and increases E-cadherin-dependent cell adhesion, J.Biol.Chem., 281: , He,T.C., Sparks,A.B., Rago,C., Hermeking,H., Zawel,L., da Costa,L.T., Morin,P.J., Vogelstein,B. and Kinzler,K.W. Identification of c-myc as a target of the APC pathway, Science, 281: ,

4 Supplemental Figures Legends Supplemental figure S1 A. Axin2 mrna expression in 2AAF/PHx-treated rats. Results are expressed as fold increase over basal value obtained in normal rat livers (three experiments). B. RT-PCR analysis of CK-19, AFP, and albumin mrna in parenchymal cell fraction (P) and nonparenchymal cell fraction (NP). C. Western blot analysis of total -Catenin and Cyclin D1 in parenchymal cell fraction (P) and nonparenchymal cell fraction (NP). D. Imunohistochemical staining for -Catenin in liver tissue obtained from 2AAF/PHx-treated rats (200x, inserted image 400x, scale bar =50 m). Arrows and arrowheads point to cytoplasmic and nuclear -Catenin staining respectively. Supplemental figure S2 A. pgl3-ot/of luciferase reporter assays of WB-F344 cells infected with Ad-blank or Ad- -catenin (S37Y) (*p<0.05). B. Cell growth curve of the Ad-blank or Ad- -catenin (S37Y)-infected WB cells. Cells were counted after trypsin digestion at different time points (*p<0.05). Supplemental figure S3 pgl3-ot/of luciferase reporter assays of Huh7 and SMMC7721 cells treated with BIO or MeBIO (*p<0.05, **p<0.01). Supplemental figure S4 Western blot analysis of total -Catenin in SMMC7721 and Huh7 cells infected with LV-mi- -catenin or LV-control. Supplemental figure S5 Reduction of percentage of OV6 + Huh7 cells after LV-mi- -catenin transduction but not after LV-control transduction (*p<0.05). 4

5 Supplemental Table 1 Primer Sequences Used for RT-PCR (Rat) Gene Name -actin ALB AFP CK19 Axin 2 Primer Sequences 5 - CGTAAAGACCTCTATGCCAA AGCCATGCCAAATGTCTCAT GCCCTACCCACAAAGCCTCAG GTGGCTTTCTGTTGCTGTTCA CATACGAAGAAAACAGGGCG GATGGTGGGTAGTTTGCAGC GGGGAGCTGGTGGTTAAGATC GCTCAGTCACAAGTTCCACCT CAGGACCCACATCCTTCT ACGCGGAGGTGCACGCGG -3 5

6 Supplemental Table 2. (Human) Primer Sequences Used for RT-PCR Gene Name 18S AFP ALB ABCG2 GATA6 C/EBP C/EBP c-kit EpCAM Notch 1 Bmi 1 Nanog Oct 4 Primer Sequences 5 -CGGCTACCACATCCAAGGAA-3 5 -GCTGGAATTACCGCGGCT-3 5 -GTTGCCAACTCAGTGAGGAC-3 5 -GAGCTTGGCACAGATCCTTA-3 5 -TTGGAAAAATCCCACTGCAT CTCCAAGCTGCTCAAAAAGC -3, 5 - CACCTTATTGGCCTCAGGAA CCTGCTTGGAAGGCTCTATG-3 5 -GCCAACTGTCACACCACAAC-3 5 -TGGAGTCATGGGAATGGAAT-3 5 -TCACCGCTCCAATGCCTAC-3 5 -CCCTATGTTTCCACCCCTTTC-3 5 -CTTCAGCCCGTACCTGGAG-3 5 -GGAGAGGAAGTCGTGGTGC-3 5 -GATGACGAGTTGGCCCTAGA-3 5 CAGGTAGTCGAGCGTTTCCT-3 5 -CTGGCCGTAAACTGCTTTGT-3 5 -AGCCCATCATTGTTCTGGAG GGCCACCTGGGCCGGAGCTTC GCGATCTGGGACTGCATGCTG GCAGCAATGACTGTGATG AGTCCATCTCTCTGGTGAC GTCCCGGTCAAGAAACAGAA TGCGTCACACCATTGCTATT GTTCAGCCAAACGACCATCT TTGCCTCTCACTCGGTTCTC-3 6

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