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

HEP_24647_sm_SuppFig1.tif652KSupplementary Figure 1. Schematic representation of the procedures for the antitumor effect of HNF1α in mice. Hep3B was inoculated into the armpits of BALB/c nude mice to generate the subcutaneous HCC model (A). AdHNF1α or AdGFP was injected intratumorally three weeks later. The animals were sacrificed after three weeks. Orthotopic liver cancer model was established in NOD/SCID mice by direct injection of Hep3B cells into liver in situ (B). Adenovirus was injected via tail vein twice a week up to 3 weeks beginning at 10d after cell inoculation. Mice were sacrificed 1 week after final virus delivery.
HEP_24647_sm_SuppFig2.tif3829KSupplementary Figure 2. Ectopic expression of HNF1α in hepatoma cells. Real-time RT-PCR (A, B), Western blot (C), and immunocytofluorescent staining (D) were performed to detect the ectopic expression of HNF1α after adenovirus-mediated HNF1α gene transfer to hepatoma cells Hep3B, Huh7, MHCC-H and MHCC-L for 3 d. The mRNA and protein expression levels were normalized against β-actin. The hepatoma cells were immunostained for HNF1α (red), GFP (green), with 4′,6-diamidino-2-phenylindole (DAPI) counterstaining for DNA (blue). The result showed enhanced expression of HNF1α was mainly located in the cell nucleus (D). The transcription of HNF1α in HNF1α-expressing adenovirus-infected HCC cell lines was compared with normal human mature hepatocytes (E). Each value represents the mean ± SD for triplicate samples. **P<0.01, ***P<0.001.
HEP_24647_sm_SuppFig3.tif2196KSupplementary Figure 3. HNF1α suppresses proliferation of hepatoma cells. Proliferation of Huh7(A, B), MHCC-H(C, D) and MHCC-L(E,F) was detected daily after infected with adenovirus. The proliferation of cells was significantly suppressed by HNF1α delivery in a time-dependent manner. Each value represents the mean ± SD for triplicate samples. ** P<0.01.
HEP_24647_sm_SuppFig4.tif2631KSupplementary Figure 4. HNF1α inhibits colony formation ability of hepatoma cells. Colonies formation of hepatoma cells Huh7(A, B), MHCC-H(C, D) and MHCC-L(E,F) was detected after virus infection. HNF1α delivery robustly inhibited the colony formation of Huh7, MHCC-H and MHCC-L.
HEP_24647_sm_SuppFig5.tif317KSupplementary Figure 5. HNF1α decreased percentage of CD133+ cells. Flow cytometry analysis was performed to measure the effect of HNF1α on the expression of CD133 in Hep3B.
HEP_24647_sm_SuppFig6.tif2379KSupplementary Figure 6. HNF1α inhibits tumorigenic cell signaling pathways. The gene expression fold in HNF1α group versus GFP group was analyzed in Hep3B(A, C), Huh7(B) after infection with adenovirus for 3d, including components of mTOR and TGFβ / smads signaling pathway, such as platelet-derived growth factor alpha polypeptide (PDGFA), platelet-derived growth factor beta polypeptide (PDGFB), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), eukaryotic translation initiation factor 4 gamma 3 (EIF4G3), eukaryotic translation elongation factor 1 alpha 2 (EEF1A2), TGFβ, TβR1, TβR2, Smad1, Smad2 and Smad5. Western blot (D) was carried out to examine the activation of TGFβ / smads pathway in Hep3B. Expression of β-actin mRNA and protein was used as an internal control. Each value represents the mean ± SD for triplicate samples. * P<0.05, ** P<0.01.
HEP_24647_sm_SuppFig7.tif2057KSupplementary Figure 7. Effect of HNF1α on cell cycles in MHCC-H and MHCC-L. Flow cytometry analysis was used to detect the effect of HNF1α on cell cycle in MHCC-H (A) and in MHCC-L (B) after virus infection for 3d. The result showed HNF1α treatment significantly elevated the percentage of cells in G2/M phase. Each value represents the mean ± SD for triplicate samples. * P<0.05.
HEP_24647_sm_SuppFig8.tif1339KSupplementary Figure 8. Effect of HNF1α on apoptosis of hepatoma cells. Apoptosis of hepatoma cells were measured by flow cytometry analysis 3d after virus infection. No apoptosis was induced by AdHNF1α in hepatoma cells except MHCC-L. Each value represents the mean ± SD for triplicate samples. ** P<0.01.
HEP_24647_sm_SuppFig9.tif669KSupplementary Figure 9. miR-192 and miR-194 inhibitors partly attenuate the suppressing effect of HNF1α on the cell proliferation. 1d after Huh7 infected with adenovirus, the cells were transfected with miR-192 or miR-194 inhibitors. Proliferation was detected daily. The proliferation of cells was significantly suppressed by HNF1α delivery, whlie this effect was partly eliminated by the inhibitor of miR-194 or miR-192 in a time-dependent manner. Each value represents the mean ± SD for triplicate samples. * P<0.05.
HEP_24647_sm_SuppFig10.tif869KSupplementary Figure 10. Effect of miR-192 and miR-194 mimics on colony formation ability of Huh7. Colony formation ability was significantly inhibited after miR-192 or miR-194 mimics transfected into Huh7. * P<0.05.
HEP_24647_sm_SuppFig11.tif747KSupplementary Figure 11. Effect of HNF1α on tumours′ differentiation in vivo. Real-time RT-PCR was taken to clarify expression of the liver-specific genes and CSCs marks in liver tissues isolated from subcutaneous HCC model. A cluster of liver-specific genes expressions were upregulated, and expression of CD133 and EpCAM decreased in AdHNF1α intratumoral injected subcutaneous xenograft models.
HEP_24647_sm_SuppFig12.tif4228KSupplementary Figure 12. Effect of HNF1α on proliferation and tumor vascularization in vivo. Immunohistochemical staining of PCNA, Ki67, CD31 and CD34 was carried out to measure tumor cell proliferation and tumor vascularization in orthotopic liver cancer model. Up-regulation of HNF1α and down-regulation of PCNA, Ki67, CD31 and CD34 were observed in HNF1α group compared with GFP group in the paraffin embedded liver sections. Scare bars, 100μm.
HEP_24647_sm_SuppInfo.doc169KSupporting Information

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