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

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HEP_26159_sm_SuppFig1.tif15888KSupplementary Figure 1. Representative double immunostaining of EpCAM/CK19, SALL4/EpCAM and SALL4/CK19 expression in human normal livers. Fetal (16 weeks gestation) and neonatal (4 months) livers (A). (a, b) EpCAM and CK19 expression in fetal (a) and neonatal (b) livers. Sections were stained with anti-EpCAM and anti-CK19 antibody. Expression of EpCAM (green) or CK19 (red) was visualized with Alexa488- and Alexa546-conjugated antibodies. Nuclei (blue) were stained with DAPI. (c, d) SALL4 and EpCAM expression in fetal (c) and neonatal (d) livers. (e, f) SALL4 and CK19 expression in fetal (e) and neonatal (f) livers. Sections were stained with anti-SALL4 and anti-EpCAM or anti-CK19 antibody. Expression of SALL4 (brown) or EpCAM, CK19 (red) was visualized with 3,3'-diaminobenzidine and Warp Red. Arrows showed ductal plate cells, now recognized to be hHpSCs, and arrowheads indicated hHBs respectively. Magnification ×400. BD, bile duct; DP, ductal plate; PT, portal tract. (B) SALL4 expression in cultures of hBTSCs. The colony was stained with antibodies against EpCAM and SALL4. Expression of EpCAM (green) or SALL4 (red) was visualized with Alexa488- and Alexa546-conjugated antibodies. Nuclei (blue) were stained with DAPI. Magnification ×400.
HEP_26159_sm_SuppFig2.tif19918KSupplementary Figure 2. Representative immunostaining of SALL4 in non-cancerous liver and HCC specimens (A-D). (A) chronic hepatitis, (B) liver cirrhosis, (C) SALL4-negative HCC (T7, well-differentiated) and (D) SALL4-positive HCC (T48, well-differentiated). Sections were stained with anti-SALL4 antibody. Magnification ×200. BD; bile duct. (E, F) Representative double immunostaining of SALL4/EpCAM and SALL4/CK19 expression in CC specimens (T5, poorly-differentiated). Serial sections were stained with anti-SALL4, anti-EpCAM and anti-CK19 antibody. Magnification ×400.
HEP_26159_sm_SuppFig3.tif598KSupplementary Figure 3. Over-expression of SALL4 proteins in cultures derived from Huh7 and PLC/PRF/5 cells. Cells were infected with mock- or SALL4-expressing viruses and stained with anti-SALL4 antibody after fixation. SALL4 expression was detected immunocytochemically. Expression of SALL4 (red) was detected using Alexa546-conjugated antibody. Nuclei (blue) were stained with DAPI.
HEP_26159_sm_SuppFig4.tif122KSupplementary Figure 4. Efficiencies of lentiviral transduction into Huh7 and PLC/PRF/5 cells. Transduction efficiency was estimated using flow cytometer (EGFP expression). The percentages of Huh7 cells infected with lentiviruses expressing-shRNA against luciferase or SALL4 were respectively 97.0%, 97.9% and (upper panel). The percentages of PLC/PRF/5 cells infected with lentiviruses expressing-shRNA against luciferase or SALL4 were respectively 94.2%, 97.6% and (lower panel).
HEP_26159_sm_SuppFig5.tif7560KSupplementary Figure 5. Morphological features of Huh7 and PLC/PRF/5 cells infected with control- and SALL4 knockdown-shRNA viruses. Cells were infected with lentiviruses expressing-shRNA against luciferase or SALL4 and cultured for 7 days. Cells infected with lentiviruses were detected by expression of EGFP (green). Nuclei (blue) were stained with DAPI.
HEP_26159_sm_SuppFig6.tif167KSupplementary Figure 6. Expression of hepatocytic differentiation (A) and stemness (B) genes in SALL4-over-expressing liver cancer cells. Cells were infected with a retroviral vector expressing SALL4 and cultured for 3 days. UGT2B7, HNF4α, TACSTD1, POU5F1, and CD90 mRNA expression was detected using qRT-PCR. Data are expressed as mean ± SD (triplicate samples, ***p<0.001, **p<0.01).
HEP_26159_sm_SuppFig7.tif128KSupplementary Figure 7. Expression of hepatocytic differentiation and EMT-related genes in SALL4-knockdown liver cancer cells. Cells were infected with a lentiviral vector expressing-shRNA against luciferase or SALL4. UGT2B7, HNF4α, TACSTD1, POU5F1, and CXCR4, TWIST1, CDH1 mRNA expression was detected using qRT-PCR. Data are expressed as mean ± SD (triplicate samples, *p<0.05).
HEP_26159_sm_SuppFig8.tif5472KSupplementary Figure 8. Representative immunostaining of SALL4 in HCC specimens (T37, moderately-differentiated) by using citrate buffer pH 6.0 (A) or EDTA buffer pH 8.0 (B) for antigen retrieval. Serial sections were stained with anti-SALL4 antibody. Magnification ×200.
HEP_26169_Supporting_Tables.doc194KSupplementary Tables

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