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

HEP_25564_sm_SuppFig1.tif1484KSupporting Information Figure 1. ZNF191 siRNA resistant cDNA recovered the expression of β-catenin and cyclin D1 proteins suppressed by ZNF191 siRNA. (A) pCMV-Myc-ZNF191SR (bearing ZNF191 siRNA resistant cDNA that contained silent mutations in the recognition site of Si-ZNF191) restored ZNF191 protein expression. Western blot analysis of ectopic expression of ZNF191 and β-actin (loading control) in L02 (left) and Hep3B (right) cells cotransfected with control siRNA (Scram-si) or ZNF191 siRNA (Si-ZNF191), and pCMV-Myc-ZNF191SR or control pCMV-Myc-ZNF191 vectors. The proteins expression was checked on day 2 after transfection. (B) pCMV-Myc-ZNF191SR recovered the expression of β-catenin and cyclin D1 proteins suppressed by ZNF191 siRNA. Western blot analysis of ZNF191, β-catenin, cyclin D1 and β-actin in L02 (left) and Hep3B (right) cells cotransfected with control siRNA or ZNF191 siRNA, and pCMV-Myc-ZNF191SR and mock pCMV-Myc vectors.
HEP_25564_sm_SuppFig2.tif761KSupporting Information Figure 2. Supporting Fig. 2. Correlation analysis between ZNF191 and β-Catenin target gene c-Myc in the 44 human HCCs. Scatter plot showing the correlation of ZNF191 with c-Myc and the non-target gene of the Wnt pathway, STAT4 (signal transducer and activator of transcription 4). Data were analyzed using the paired t-test, and correlation analysis was performed using Pearson's correlation test.
HEP_25564_sm_SuppFig3.tif3215KSupporting Information Figure 3. No mutations of exon 3 region of human β-Catenin gene were found in all 44 HCC tumors. Representative of sequencing chromatogram of the exon 3 region of β-Catenin gene in a HCC tumor examined, and the underlying characters indicate the codon 32-45 in exon 3. Similar results were detected in other 43 HCC tumors.
HEP_25564_sm_SuppFig4.tif8228KSupporting Information Figure 4. ZNF191 cannot directly bind to the CCND1 promoter (-962CD1). (A) β-catenin and ZNF191 containing DNA-protein complexes were immunoprecipitated as verified by immunoblotting with the corresponding antibody: anti-myc (Sigma-Aldrich), polyclonal anti-β-catenin (Sigma-Aldrich), anti-ZNF191(Abcam). Mouse IgG or Rabbit IgG (Sigma-Aldrich) were used as a negative control. (B) ZNF191 cannot direct bind to LEF/TCF site of the CCND1 promoter, while β-catenin directly binds to the site. (C) ZNF191 cannot direct bind to indicated CCND1 promoter regions (covered full length of -962CD1). In vivo ChIP assays were performed on Hep3B samples and PCR reactions were performed using specific primers listed in Supporting Table 2.
HEP_25564_sm_SuppFig5.tif649KSupporting Information Figure 5. Purification of recombinant ZNF191. SDS-PAGE of recombinant ZNF191. Size ladder (PM, protein marker), cell extract without IPTG (Lane 1), cell extract induced by IPTG (Lane 2), purified ZNF191 at approximately 48 KD (Lane 3, 4, 5, loading 2, 4, 6μl, respectively)
HEP_25564_sm_SuppTabInfo.doc131KSupporting Information Tables.

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