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

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HEP_25914_sm_SuppFig1.tif4745KSupporting Information Figure 1. Figure S1. Liver-specific inducible transgenic zebrafish. A, The liver-specific activator plasmid pT2-LF-tTA (a) containing an activator (tTA) driven by a zebrafish L-FABP promoter, and the tetracycline-responsive plasmids pT2-GFP-BI-HCP, pT2-GFP-BI-HBx, pT2-HcRed-BI-HBx, and pT2-GFP-BI (b-e, respectively) containing genes driven by a Tet-controlled bidirectional promoter, are illustrated. All expression cassettes were flanked by the Tol2 transposon. B, Fluorescence photomicrograph of 3-month-old transgenic zebrafish. C, The amount of viral mRNA in individual samples was estimated using RT-PCR with the HBx and HCP primers.
HEP_25914_sm_SuppFig2.tif3021KSupporting Information Figure 2. Whole mount in situ hybridization of 5 dpf zebrafish lines with HBx and HCP antisense probes (HBx-AS and HCP-AS, respectively; sense probe HBx-S and HCP-S as control). No signal was observed in HCP and HBx with HBx-AS and HCP-AS, respectively.
HEP_25914_sm_SuppFig3.tif1649KSupporting Information Figure 3. Immunofluorescence assay of CT zebrafish. The bile duct and nucleus of CT liver showed positive staining for 2F11/2 (red) and DAPI (blue), respectively. GFP (green) was not localized in the 2F11/2-positive cells.
HEP_25914_sm_SuppFig4.tif3976KSupporting Information Figure 4. Gross and fluorescent morphology of 3-month-old HBx+HCP zebrafish. ICC in HBx+HCP zebrafish displayed darkly pigmented regions (A) without a fluorescence signal (B) within the liver compared with a CT fish liver (C and D).
HEP_25914_sm_SuppFig5.tif3049KSupporting Information Figure 5. Figure S5. Histological examination of transgenic zebrafish livers. None of the zebrafish carrying a single viral gene or the vector had ICC formation at 3 months of age. The arrow indicates the bile duct.
HEP_25914_sm_SuppFig6.tif2541KSupporting Information Figure 6. Figure S6. Histological examination of ICC that was induced by conditional co-expression of HBx and HCP. Liver of HBx+HCP transgenic zebrafish with 3 (A) and 1.5 (B) months of Dox treatment showing no obvious alterations. (C) zebrafish subjected to 3 month-withdrawal showing ICC formation.
HEP_25914_sm_SuppFig7.tif3943KSupporting Information Figure 7. Figure S7. Evaluation of proliferation by PCNA immunostaining in the liver of transgenic zebrafish lines. Liver of HBx (A) and HCP (B) zebrafish showing a very low proliferation index. Liver of HBx and HCP zebrafish with dilated bile ducts showing increased proliferation (C), whereas ICC showing a very high proliferation index (D).
HEP_25914_sm_SuppFig8.tif3882KSupporting Information Figure 8. Figure S8. Immunohistochemical in situ TUNEL detection of apoptosis in zebrafish liver. Liver of HBx (A) and HCP (B) zebrafish showing a low DNA damage. Liver of HBx and HCP zebrafish with dilated bile ducts showing a low DNA damage (C), whereas ICC showing a high DNA strand breaks in the tissues surrounding the abnormal bile ducts (D).
HEP_25914_sm_SuppFig9.tif1900KSupporting Information Figure 9. Figure S9. Co-expression of HBx and HCP increased the expression of pSmad3L oncogenic pathway-related genes. The expression levels of the TGF-?1- and pSmad3L-related genes (tgfb1, smad2, smad3, p38, erk1, kras, ctgf, ccnd1, and vegfa) involved in ICC formation were examined using quantitative RT-PCR and transcriptome sequencing (RNAseq) in 3-month-old zebrafish livers. The expression level of gapdh served as an internal control. The qRT-PCR experiment was performed in biological triplicate. The error bars indicate the S.D.
HEP_25914_sm_SuppFig10.tif4397KSupporting Information Figure 10. Figure S10. Immunohistochemistry of HBx and HCP transgenic zebrafish liver for TGF-?1, ?-SMA, and pp38. Weak TGF-?1 was detected in the livers isolated from HBx or HCP transgenic zebrafish. The liver tissue showing ?-SMA immunostaining surround the bile duct in HBx zebrafish. The liver tissue showing weak immunostaining of phosphorylated p38 (pp38) in HBx or HCP transgenic zebrafish. Arrow indicates the bile duct.
HEP_25914_sm_SuppFig11.tif4405KSupporting Information Figure 11. Figure S11. Immunohistochemistry of HBx and HCP transgenic zebrafish liver for pERK1/2, pSmad3L, and CK18. Weak pERK1/2, pSmad3L, and CK18 were detected in the livers isolated from HBx or HCP transgenic zebrafish. Arrow indicates the bile duct.
HEP_25914_sm_SuppFig12.tif801KSupporting Information Figure 12. Figure S12. Injection of tgfb1-MO suppressed the pSmad3L oncogenic pathways in the liver of HBx+HCP zebrafish. A, RT-PCR was performed to confirm that tgfb1 expression was suppressed in morphants. B, The liver samples of the 3-month-old morphants were immunoblotted with antibodies against TGF-?1, phospho-Smad3L, phospho-p38, phospho-ERK1/2, and GAPDH (internal control). C, The expression levels of the TGF-?1-related genes (tgfb1, smad2, smad3, p38, and jnk) and pSmad3L downstream gene (mmp9) were examined using quantitative RT-PCR in the liver of tgfb1 morphants. *, p < 0.05, t test. The error bars indicate the S.D.
HEP_25914_sm_SuppTab1to4.doc95KSupporting Information Table 1 to 4.
HEP_25914_sm_SuppTab5.xls7605KSupporting Information Table 5. The differential expression data of transgenic zebrafish samples (continue).
HEP_25914_sm_SuppTab6.xls251KSupporting Information Table 6. The gene list of the top-ranked up-regulated genes more than 2-fold and down-regulated genes more than 0.5-fold changes identified by differential expression analysis followed by pathway analysis (MetaCore).
HEP_25914_sm_SuppInfo.doc44KSupporting Information

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