1. Volume 133, Issue 5, Pages 1579-1591.e1 (November 2007)
Wnt/β-Catenin Signaling in Murine Hepatic Transit Amplifying Progenitor Cells 
Min Hu, Masashi Kurobe, Yeon Jun Jeong, Christophe Fuerer, Saif Ghole, Roel Nusse, Karl G. Sylvester 
Gastroenterology 
Volume 133, Issue 5, Pages e1 (November 2007)
DOI: /j.gastro
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2. Figure 1
(A) H&E of DDC-stimulated liver at 3 weeks, showing structural changes accompanying oval cell proliferation and ADP formation (arrowheads) at the PT (200×) (asterisks demarcate porphyrin accumulation). (B) PCNA-positive oval cells in ADP and surrounding small hepatocytes (200×). (C) Absence of the mitotic nuclear antigen P-H3 staining in noninjured liver (200×). (D) P-H3 staining localizes to oval cells within the ADP (arrowheads) in the PT area of DDC-stimulated livers (asterisks demarcate porphyrin accumulation) (400×). (E) Immunohistochemical detection of β-catenin in noninjured control livers is limited to the plasma membrane of hepatocytes and sinusoidal endothelium (200×). (F) Localization of β-catenin in oval cells within the ADP of DDC-stimulated livers. The arrowheads show areas of nuclear β-catenin. β-Catenin localization in surrounding hepatocytes is largely membranous (400×).
Gastroenterology  , e1DOI: ( /j.gastro )
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3. Figure 2
(A) Low power of A6 and β-catenin coimmunolocalization in 3 week DDC-stimulated mouse liver (100×). Scale bar is 50 μm. (B) The A6 antigen in red localizes to ADP oval cells (200×). (C) β-catenin within ADP oval cells and membranous staining in hepatocytes (200×). (D) Higher magnification merged image of panels B and C, showing the overlapping staining pattern (yellow) of A6 and β-catenin (630×).
Gastroenterology  , e1DOI: ( /j.gastro )
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4. Figure 3
β-Galactosidase (β-gal) histochemical staining, indicating TCF/LEF reporter activity in mouse liver. (A) In noninjured livers during homeostasis β-gal activity is limited to the centrilobular hepatocytes surrounding the CV (400×). (B) Minimal β-gal reporter activity is detectable at the PT in control noninjured liver (200×). (C) Increased β-gal activity in ADP oval cells surrounding the PT in DDC-treated livers (100×). (D) Higher magnification view of a PT after DDC stimulation, showing β-gal staining in ADP oval cells, indicating TCF/LEF reporter activation (400×).
Gastroenterology  , e1DOI: ( /j.gastro )
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5. Figure 4
Wnt family gene induction by quantitative PCR and their localization by in situ hybridization in DDC-treated livers. Each of the bar graphs for the indicated Wnt transcripts represent the relative mRNA expression value for DDC treated (black bars) and control livers (empty bars). Each of the relative differences depicted reached statistical significance (P < .05). Values are mean ± SD. Above each bar graph for the indicated Wnt gene is the localization of the corresponding mRNA by in situ hybridization within DDC-treated livers at the same 3-week time point. Low power (200×) views and high power (630×) insets are shown. The chromagen for in situ hybridization is 5-bromo-4-chloro-3-indolyl-phosphate/4-nitro blue tetrazolium that demarcates cells with a positive signal by blue to purple colorization.
Gastroenterology  , e1DOI: ( /j.gastro )
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6. Figure 5
Analysis of MACS-depleted cells before culture. FACS analysis of the NP fraction of cells stained for CD45 before depletion by MACS (B) and after depletion (C). (A) An analysis of the cells without CD45 antibody is depicted. The percentage of CD45 cells in the NP fraction (B; 34.8%) is significantly reduced by MACS (C; 3.99%). The MACS-depleted cells from panel C were cytospun and stained for A6 (D) and PH3 (E) before culture, showing a highly enriched population of oval cells.
Gastroenterology  , e1DOI: ( /j.gastro )
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7. Figure 6
Results of oval cell isolation and characterization. (A) Phase contrast and differential interference contrast photomicrograph, depicting the shape of oval cells in culture (200×). (B) Candidate hepatic transcription factors present in the isolated oval cell population from DDC-stimulated mouse liver. (C) Immunofluorescence detection of the oval cell marker antigen AFP in isolated oval cells in culture (200×). (D) Double immunofluorescence for β-catenin (green) in A6+ (red) cells in vitro (100×).
Gastroenterology  , e1DOI: ( /j.gastro )
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8. Figure 7
(A) Immunofluorescent localization of β-catenin nuclear translocation in response to Wnt3a treatment (48 hours). (B) TCF/Luciferase (MEGATop) assay confirming significant β-catenin/TCF pathway activation in response to Wnt3a stimulation (48 hours) of oval cells (*P < .0001).
Gastroenterology  , e1DOI: ( /j.gastro )
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9. Figure 8
Results of Wnt3a treatment of oval cells in vitro. (A) Wnt3a stimulation results in statistically significant oval cell cell-cycle entry as measured by BrdU incorporation (*P < .001). (B) Wnt3a stimulation of oval cells results in a significant induction of dephosphorylated (active) β-catenin after serum starvation either in the presence or absence of serum. (C) The reporter LSL cell line confirms the biologic activity of Wnt3a protein in vitro and in the absence of serum (D). Immunoprecipitation (IP) for total β-catenin in proliferating oval cells in vitro. The Wnt-activated amino-terminal dephosphorylated (Thr41/Ser45) form of β-catenin predominates over the tyrosine phosphorylated (Tyr142/654) form of β-catenin.
Gastroenterology  , e1DOI: ( /j.gastro )
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