Additional Supporting Information may be found in the online version of this article.

HEP_24156_sm_suppinfo.doc35KSupporting Information
HEP_24156_sm_suppinfofigure1.tif635KSupporting Figure 1. Up-regulation of Sonic Hedgehog ligand mRNA in children with biliary atresia and other types of cholestatic liver disease. Patients with BA, children with various other cholestatic liver diseases, and age-matched controls without liver disease (nondiseased, ND), were examined for Shh ligand expression. QRT-PCR analysis was performed in liver tissue from patients with BA (N=9), Alagille's syndrome (AGS, N=5), progressive familial intrahepatic cholestasis type 1 (FIC1, N=7), progressive intrahepatic cholestasis type 2 (BSEP, N=6), and age-matched CTL (N=6). (A) Shh ligand mRNA expression. Gene expression data in diseased livers are expressed relative to levels in control subjects and graphically depicted using box-and-whisker plots. As evaluated by Wilcoxon rank-sum test, no significant differences in Shh expression were noted among the groups.
HEP_24156_sm_suppinfofigure2.tif4068KSupporting Figure 2 Strong correlation between expression of Snail and that of other mesenchymal markers in BA patients. (A-C) Covariance between the EMT-driving gene, Snail, and two other typical mesenchymal markers, Vimentin and FSP1, was evaluated in BA patients. Data were plotted to demonstrate the results of linear regression analysis. Significance (P=value) and strength of correlation (coefficient of determination, r2) are shown. (D-E) Pictures demonstrating FSP1 staining in the liver (intrahepatic, D) and extrahepatic biliary remnant (E) of representative BA patients. Magnification 200×.
HEP_24156_sm_suppinfofigure3.tif12458KSupporting Figure 3 Hh-responsive (Gli2-positive) cells co-express mesenchymal and epithelial markers in BA livers and extrahepatic biliary remnants. Immunohistochemical analysis for Gli2/Vimentin (A-B) and Gli2/KRT7 (C-D) was performed in both livers (A, C) and extrahepatic biliary remnants (B, D) from BA patients. Final magnifications 100×
HEP_24156_sm_suppinfofigure4.tif6565KSupporting Figure 4 Progenitor marker expression in BA livers and extrahepatic biliary remnants. Immunohistochemical analysis for AE1/AE3 (A-B) and Epcam (C-D) was performed in both livers (A, C) and extrahepatic biliary remnants (B, D) from BA patients. Final magnifications 200×
HEP_24156_sm_suppinfofigure5.tif1655KSupporting Figure 5. FSP1 expression is Hedgehog-regulated in normal primary rat cholangiocytes. QRT-PCR (A) and immunocytochemistry (B) of pooled small and large primary cholangiocytes from healthy adult rats were used to assess FSP1. (A) FSP1 mRNA expression after treatment with unconditioned medium (white bars) containing control IgG or MF-conditioned with control IgG (black bars) or Hh neutralizing antibody (grey bars). (B) Representative cholangiocyte cytospins. Final Magnification 400×, inserts show magnified field of areas pointed by the arrows. (C) Quantification of FSP1 positive cholangiocytes. FSP1(+) (red bars) and FSP1 (-) (black bars) cholangiocytes were counted under under 200× final magnification. Per each field, FSP1 positive (i.e. red (+) and DAPI(+)) (red bars) and FSP1 negative (i.e. red (-) and DAPI(+)) were counted and the final data were expressed as percentage of total cells/field. (D) QRTPCR analysis of FSP1 expression in small (more immature) and large (mature) subpopulations of primary cholangiocytes isolated from rats with ongoing biliary fibrosis (i.e. BDL). Data are expressed relative to control group (white bars) and displayed as mean ± Standard Error Mean (SEM). Comparisons between groups were performed using the two-tailed Student's t-test. Significance was accepted at the 5% level. (*P<0.05, ** P<0.005).
HEP_24156_sm_suppinfotable1.doc38KSupporting Table 1. Antibodies and retrieval techniques used for IHC.

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