Supporting InformationLee et al. 10.1073/pnas.0912203107SI Materials and MethodsGeneration of WW45 Liv-cKO Mice and Genotyping. TheWW45 locuswas isolated from a 129/Sv mouse BAC library. A 6.3-kb EcoRIfragment from the 5′ region of theWW45 genomic DNA was clonedinto the EcoRI site of pBluescript(+) KS (Stratagene), and a phos-phoglycerate kinase genepromoter–neomycin resistance gene (neo)–poly(A) cassette containing both LoxP and FRT sites was insertedinto the EcoRI-BamHI site in the reverse orientation. A 3.4-kbBamHI fragment was then cloned into the 3′ end of the targetingvector. The targeting vector also contained a LoxP site in the intronpreceding exon 2 in addition to the floxed neomycin resistance cas-sette in the intron downstream of exon 2. The targeting vector wasintroduced into mouse ES cells, and homologous recombination wasconfirmedbySouthernblot analysisof genomicDNAwithanexternalprobe. Genotyping of mice was performed by PCR analysis with theprimers ww-L2 (5′-TGGTTTGCTTTTTAGTGGCC-3′) andww-R3(5′-TGCTGGTTTTGTCTCACTAA-3′), which yield products of158 and 238 bp for the WT and floxed alleles, respectively. Micehomozygous for the floxed allele were viable, grew at a normal rate,andwere fertile and phenotypically normal. For selective inactivationof the WW45 gene in the liver, the WW45flox/flox mice were crossedwithAlbumin-Cre transgenicmice, which express Cre recombinase inthe postnatal liver including hepatocytes and oval cells (1). Re-combination PCR analysis with the primers ww-L3 (5′-CTTATG-CCCTTTTGTTTGAT-3′) andww-R3, which yield products of 1,333and 165 bp for the undeleted and excised alleles, respectively, con-firmed the tissue specificity of Cre-mediated gene disruption (2).WW45flox/flox mice were used as controls in this study. Genotyping ofhepatomas that developed inWW45+/−mice was performed by PCRanalysis with the primers as previously described (3).

Partial Hepatectomy and Analysis. Eight-week-old mice were anes-thetized by ether inhalation and subjected to two-thirds partialhepatectomy as previously described (4). Six different microscopicfields (magnification, ×200) were randomly selected for quantifi-cation of BrdU-positive cells in liver sections immunostained forBrdU and stained with DAPI. All experiments were performed incompliance with the standards for animal use and care of theKoreaAdvanced Institute of Science and Technology.

Histologic Analysis. Sections (4 μm) were stained with H&E or weresubjected to immunohistochemical analysis according to standardprotocols. Antibodies for immunostaining included those to albu-min (Dako), toα1-antitrypsin (Invitrogen), to pan-cytokeratin (pan-CK) (Dako), to proliferating cell nuclear antigen (PCNA) (SantaCruz Biotechnology), to BrdU (BD Bioscience), to Ki67 (Novo-castra), to A6 (kindly provided by V. Factor), and to YAP (CellSignaling) as well as FITC-conjugated anti-CD34 (eBioscience).Peroxidase activity was detected with an EnVision+ Dual LinkSystem HRP (DAB+; Dako). The TUNEL assay was performedwith the use of an In Situ Cell Death Detection Kit (Roche).For detection of BrdU incorporation in vivo, mice were injected

i.p. with BrdU (Sigma) at a dose of 100 μg per gram of body weightand 2 h before analysis. Quantitation of staining was performedwith NIH ImageJ software (http://rsb.info.nih.gov/ij/). For quan-tification of the PCNA proliferative index, six different micro-scopic fields (magnification, ×200) were randomly selected forcounting of cells stained for PCNA and pan-CK; the percentage of

cells negative or positive for pan-CK that were also positive forPCNA was determined.

Isolation and Analysis of Hepatocyte and Oval Cell–Enriched Fractions.Hepatocytes and nonparenchymal cells were isolated by collage-nase digestion of the liver tissue, and the hepatocyte fraction wastheneliminatedby twoconsecutivecentrifugations for5minat50×g. The final supernatant, constituting a nonparenchymal fraction,was subjected to centrifugation on a discontinuous Percoll (Sigma-Aldrich) gradient of 30% and 70% solutions, and the cell fractionthat collected at the interphase of the two Percoll solutions wasisolated. The cells were washed with Williams E medium supple-mented with 10% FBS and were then plated in collagen-coatedculture dishes in growth medium, consisting of high-glucoseDMEM-F12 (1:1) (HyClone) supplemented with Hepes, sodiumpyruvate, insulin, transferrin, selenium, penicillin, streptomycin,and 10% FBS. They were maintained at 37 °C in a humidifiedincubator containing 5% CO2. After culture for 1 day, non-adherent cells including lymphocytes were removed, and the en-riched oval cells were maintained. Resulting visible oval cellcolonies were subjected to immunofluorescence analysis or assayof BrdU incorporation. For immunofluorescence analysis, cellswere fixed in methanol for 20 min at −20 °C and then exposedconsecutively to primary and secondary antibodies. Slides weremounted with DAPI and imaged. For assay of BrdU in-corporation, cells were incubated for 5 h in growth medium con-taining 10 μM BrdU, fixed, and stained with antibodies to BrdUaccording to standard protocols.

Immunoblot Analysis. Antibodies for immunoblotting includedthose to YAP, to Ser127-phosphorylated YAP, toMST1, to Ser909-phosphorylated LATS1 (Cell Signaling), to LATS1, to LATS2(Bethyl Laboratories), to cyclin D1, to lamin B (Santa Cruz Bio-technology), to α-tubulin (Chemicon), to β-actin (Sigma), toGAPDH (Abcam), or to WW45 (rabbit polyclonal, Ctr#1) (3).

Blood Serum Analysis. Blood serum was analyzed with the use ofa Hitachi 7180 biochemical analyzer.

Quantitative RT-PCR Analysis. RNA preparation and cDNA syn-thesis were performed as previously described (5). QuantitativeRT-PCR analysis was performed with the primers Ctgf-F (5′-GGGCCTCTTCTGCGATTTC -3′) and Ctgf-R (5′-ATCCAGG-CAAGTGCATTGGTA-3′) for CTGF; Birc5-F (5′-GAGGCTG-GCTTCATCCACTG-3′) andBirc5-R (5′-CTTTTTGCTTGTTG-TTGGTCTCC-3′) forBIRC5;Sav1-F (5′-CTGTCCCGCAAGAA-AACCAAA-3′) and Sav1-R (5′-AATGAAGGCATGAGATTCC-GC-3′) for SAV1;Yap-F (5′-TACTGATGCAGGTACTGCGG-3′)and Yap-R (5′-CAGGGATCTCAAAGGAGGAC-3′) for YAP;Mst1-F (5′-GCTGTGGTCAGACAACTTCAT-3′) and Mst1-R(5′-TGACACTCCTTTGGCACTCTTA-3′) for MST1; Mst2-F (5′-CGGGGTCCGTTTCAGACATAA-3′) andMst2-R (5′-GCGTTT-TGCCATTGTATCTGTT-3′) for MST2; and Lats2-F (5′-GGAC-CCCAGGAATGAGCAG-3′) andLats2-R (5′-CCCTCGTAGTT-TGCACCACC-3′) for LATS2. Data were normalized by theabundance of GAPDH mRNA determined with the primersGapdh-F (5′-GCACAGTCAAGGCCGAGAAT-3′) andGapdh-R(5′-GCCTTCTCCATGGTGGTGAA-3′).

1. YechoorV,etal. (2009)Neurogenin3 is sufficientfor transdeterminationofhepaticprogenitorcells into neo-islets in vivo but not transdifferentiation of hepatocytes. Dev Cell 16:358–373.

2. Postic C, Magnuson MA (2000) DNA excision in liver by an albumin-Cre transgeneoccurs progressively with age. Genesis 26:149–150.

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3. Lee JH, et al. (2008) A crucial role of WW45 in developing epithelial tissues in themouse. EMBO J 27:1231–1242.

4. Mitchell C, Willenbring H (2008) A reproducible and well-tolerated method for 2/3partial hepatectomy in mice. Nat Protoc 3:1167–1170.

5. Lee D, et al. (2008) ER71 acts downstream of BMP, Notch, and Wnt signaling in bloodand vessel progenitor specification. Cell Stem Cell 2:497–507.

Fig. S1. Generation of liver-specific WW45 conditional knockout mice. (A) Schematic representation of the targeting strategy for deletion of WW45 spe-cifically in the liver. The 5′ external probe for Southern hybridization of genomic DNA, predicted sizes of wild-type (WT) and mutant (MT) hybridizingfragments, and primers (ww-L2 and ww-R3) for PCR analysis are also indicated. Bg, BglII restriction site. (B) Southern blot analysis with the 5′ external probe ofBglII-digested genomic DNA from ES cell clones and F1 mice (Left). The positions of the WT (+, 7.5 kb) and targeted (n, 14.2 kb) alleles are indicated, with theasterisk denoting an ES cell clone positive for homologous recombination. Genomic DNA from mice was also subjected to genotyping by PCR analysis (Right).The positions of products corresponding to the WT (+, 158 bp) and floxed (f, 238 bp) alleles are indicated. (C) Recombination PCR analysis of various organs ofWW45 Liv-cKO mice (Left). The positions of products derived from the undeleted (1,333 bp) and deleted (165 bp) WW45 alleles are indicated. Liver homo-genates of WW45 Liv-cKO (cKO) and control (Ctrl) mice were also subjected to immunoblot analysis with antibodies to WW45. The position of the WW45 bandis indicated.

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Fig. S2. Ablation of WW45 in the liver promotes oval cell expansion. (A) Sections of the liver from 6-month-old control or WW45 Liv-cKO mice were stainedwith H&E (Upper). The density of nuclei in such liver sections was also determined by staining with DAPI (Lower) and examination of six randomly selectedmicroscopic fields (magnification, ×200). Data are presented as a box-and-whisker plot (control, n = 5; WW45 Liv-cKO, n = 6). (B) Immunostaining of liversections from 6-month-old control andWW45 Liv-cKO mice for the mature hepatocyte markers albumin (ALB, green) and α1-antitrypsin (AAT, red). Nuclei werealso stained with DAPI (blue). (C) Liver sections from 6-month-old control or WW45 Liv-cKO mice were subjected to TUNEL staining (green) for apoptotic cells(Upper). Nuclei were also stained with DAPI (blue). Serum from 6-month-old animals (n ≥ 6) was also assayed for ALT and AST (Lower). (D) Immunostaininganalysis of liver sections from 6-month-old control and WW45 Liv-cKO mice with FITC-conjugated antibodies to CD34 (green) and antibodies to pan-CK (red).PT, portal tract. (Scale bars, 50 μm in A; 100 μm in B–D.)

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Fig. S3. Intact regenerative capacity of hepatocytes in WW45 Liv-cKO mice subjected to partial hepatectomy. (A) Appearance of the liver of control or WW45Liv-cKO mice at the indicated times after a two-thirds partial hepatectomy (PHx) performed at 8 weeks of age. The liver was restored completely by 2 weeksafter PHx in mice of either genotype. (B) Liver weight as a percentage of body weight at 2 days and 2 weeks after partial hepatectomy in control and mutantmice (n ≥ 3). (C) Control or mutant mice (n ≥ 3) were injected i.p. with BrdU (100 μg/g) at 2 days or 2 weeks after partial hepatectomy. They were killed 2 h afterthe injection, and liver sections were subjected to immunofluorescence staining for BrdU. Sections were also stained with DAPI, and the percentage of DAPI-stained cells that were positive for BrdU was determined. (D) Liver sections prepared from mice 2 days after PHx were subjected to immunofluorescencestaining with antibodies to A6 (green) and to staining with DAPI (blue). (Scale bar, 100 μm.)

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Fig. S4. Increased proliferative response of A6-positive oval cells to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in WW45 Liv-cKO mice. (A) Liver sectionsfrom control or WW45 Liv-cKO mice fed a diet containing 0.1% DDC for 7 days were subjected to H&E staining or to fluorescence microscopy after stainingwith antibodies to A6 (green) and with DAPI (blue). A6 localizes to oval cells in atypical ductal proliferations associated with portal tracts. Asterisks demarcateporphyrin accumulation. (B) Control or mutant mice (n ≥ 3) fed a 0.1% DDC diet for 7 days were injected i.p. with BrdU (100 μg/g) and killed 2 h later. Liversections were subjected to immunofluorescence staining for BrdU and A6, and the number of A6-positive cells that were also positive for BrdU per portal tractwas determined. (C) Liver sections from control or WW45 Liv-cKO mice fed a diet containing 0.1% DDC for 2 or 3 weeks were subjected to H&E staining or toimmunohistochemical staining with antibodies to A6. Asterisks demarcate porphyrin accumulation. PT, portal tract; CV, central vein. (D) Liver sections fromcontrol or WW45 Liv-cKO mice fed a diet containing 0.1% DDC for 4 weeks were subjected to TUNEL staining (green) for apoptotic cells. Nuclei were alsostained with DAPI (blue). (Scale bars, 200 μm.)

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Fig. S5. WW45 inactivation in liver results in YAP deregulation. (A) Lysates of isolated hepatocytes and an oval cell–enriched fraction (OEF) from control (C)and WW45 Liv-cKO mice were subjected to immunoblot analysis with antibodies to the indicated proteins. (B) YAP immunofluorescence in Fig. 3E (main text)was quantitated with the use of ImageJ software and is shown according to the indicated intensity scale. (Scale bar, 50 μm.) (C) Quantitative RT-PCR analysis ofthe indicated mRNAs in isolated hepatocytes (HC) and an oval cell-enriched fraction (OEF) from control (C) and WW45 Liv-cKO mice. Gene expressions weremeasured in triplicate. Data were normalized by the amount of GAPDH mRNA and expressed relative to the normalized value for control cells. *P < 0.05.

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Fig. S6. Development of liver tumors in WW45 mutant mice. (A) Kaplan-Meier survival curves for WW45+/+, WW45+/−, and WW45−/− mice (Left) and incidenceof various tumors detected at death in WW45+/− mice (Right). The heterozygotes also developed lymphomas and osteosarcomas, albeit at lower frequenciesthan hepatomas. (B) Liver tumors in WW45+/−, WW45−/−, and WW45 Liv-cKO mice of the indicated ages. Black arrows indicate tumor nodules. (C) PCR analysisof the WT and null WW45 alleles in normal liver tissue from mice of the indicated genotypes as well as in hepatomas (T1, T2, and T3) that developed inWW45+/− mice (Left). Liver extracts of control (C) orWW45 Liv-cKO mice as well as extracts of a normal liver (+/−) and hepatomas (T1, T2, T3, and T4) ofWW45+/−

mice were also subjected to immunoblot analysis with antibodies to WW45 (Right). The position of the WW45 band is indicated. Asterisks denote nonspecificbands. (D) Quantitative RT-PCR analysis of the indicated mRNAs in nontumor and tumor tissue of the liver in WW45+/− mice (n = 4). Data were normalized bythe amount of GAPDH mRNA and expressed relative to the normalized value for nontumor tissue.

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Table S1. Blood serum analysis of control and WW45 Liv-cKOmice

Biochemical parameter Ctrl WW45 Liv-cKO

Albumin (g/dL) 1.72 ± 0.12 1.73 ± 0.11AST (U/L) 122.1 ± 62.23 116.67 ± 55.46ALT (U/L) 33.51 ± 2.89 40.73 ± 13.71ALP (U/L) 200.33 ± 21.31 210.14 ± 26.89T-cho (mg/dL) 110 ± 10.14 107.43 ± 17.05TG (mg/dL) 162.17 ± 90.36 163.71 ± 91.05T-bil (mg/dL) 0.053 ± 0.023 0.091 ± 0.025HDL (mg/dL) 68.83 ± 8.30 63 ± 11.60LDL (mg/dL) 2.67 ± 1.75 4.14 ± 1.57

Serum analysis in control and WW45 Liv-cKO mice at 6 months of age.Data are means ± SD (n ≥ 7). ALP, alkaline phosphatase; T-cho, total choles-terol; TG, triglyceride; T-bil, total bilirubin; HDL, high-density lipoproteincholesterol; LDL, low-density lipoprotein cholesterol.

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