These authors contributed equally to the study.
Version of Record online: 4 DEC 2012
Copyright © 2012 American Association for the Study of Liver Diseases
Volume 56, Issue 6, pages 2268–2276, December 2012
How to Cite
Liu, W., Chen, J.-R., Hsu, C.-H., Li, Y.-H., Chen, Y.-M., Lin, C.-Y., Huang, S.-J., Chang, Z.-K., Chen, Y.-C., Lin, C.-H., Gong, H.-Y., Lin, C.-C., Kawakami, K. and Wu, J.-L. (2012), A zebrafish model of intrahepatic cholangiocarcinoma by dual expression of hepatitis B virus X and hepatitis C virus core protein in liver. Hepatology, 56: 2268–2276. doi: 10.1002/hep.25914
Potential conflict of interest: Nothing to report.
Supported by grants from Academia Sinica, Taiwan. The Taiwan Mouse Clinic is funded by the National Research Program for Biopharmaceuticals (NRPB) at the National Science Council (NSC) of Taiwan.
- Issue online: 4 DEC 2012
- Version of Record online: 4 DEC 2012
- Accepted manuscript online: 22 JUN 2012 03:59AM EST
- Manuscript Accepted: 9 JUN 2012
- Manuscript Received: 4 SEP 2011
- Academia Sinica, Taiwan
- National Research Program for Biopharmaceuticals (NRPB) at the National Science Council (NSC) of Taiwan
Additional Supporting Information may be found in the online version of this article.
|HEP_25914_sm_SuppFig1.tif||4745K||Supporting 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.tif||3021K||Supporting 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.tif||1649K||Supporting 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.tif||3976K||Supporting 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.tif||3049K||Supporting 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.tif||2541K||Supporting 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.tif||3943K||Supporting 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.tif||3882K||Supporting 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.tif||1900K||Supporting 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.tif||4397K||Supporting 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.tif||4405K||Supporting 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.tif||801K||Supporting 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.doc||95K||Supporting Information Table 1 to 4.|
|HEP_25914_sm_SuppTab5.xls||7605K||Supporting Information Table 5. The differential expression data of transgenic zebrafish samples (continue).|
|HEP_25914_sm_SuppTab6.xls||251K||Supporting 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).|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.