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Supplementary materials and figure legends
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1. Supplementary materials and methods
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Establishment of Stable HCC Lines Expressing HCV core
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The HCV core protein coding region (genotype 1a) was PCR-amplified and
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cloned into a retroviral vector pSEB-3Flag that also conferred resistance to
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Blasticidin. The cloning junctions and PCR-amplified coding regions were
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verified by DNA sequencing. Core protein expression was determined by
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anti-FLAG antibody (Sigma, F1804). The RV-HCV core vector was transfected
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into a retroviral packaging line (empty vector as a control), and the
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recombinant retrovirus was used to infect Huh7 or SK-Hep1 cells followed by
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Blasticidin selection. The resultant stable pools were designated as Huh7-Core
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or SK-Hep1-Core. Overexpression of the core in these lines was verified by
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qPCR and western blot analysis.
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RNA isolation and quantitative RT-PCR analysis
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Total RNA were extracted from cultured cells using TRIZOL Reagents
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(Invitrogen, CA, USA) according to the manufacturer's protocol. First-strand
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cDNA synthesis was generated using random primers and MMLV RT
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(Promega, WI, USA). Polymerase chain reaction (PCR) amplifications of the
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respective genes were carried out with the cDNA products as templates. SYBR
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Green-based qPCR analysis was carried out using the DNA Engine Opticon 2
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real-time PCR detection system (Bio-Rad, CA, USA). Relative expression was
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calculated as a ratio of specific transcript to glyceraldehyde 3-phosphate
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dehydrogenase (GAPDH). Each sample was analyzed in triplicate. The primer
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sequences are listed in Supplementary Table 1.
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Western Blot analysis
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Whole cell extracts of exponentially growing cells were collected in lysis buffer
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(Promega, WI, USA) containing the complete cocktail of proteases inhibitors
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(Roche, IN, USA). Protein concentrations were determined by using the BCA
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protein assay reagent (Pierce, IL, USA). Protein samples were separated in
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10% SDS-polyacrylamide gel and electrotransferred to PVDF membranes
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(Millipore, MA, USA). The blots were probed with antibodies against HCV core
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(abcam, ab2740), HCV NS3 (abcam, ab21124), HCV E1 (abcam, ab21306),
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Flag (Sigma, F1804), SFRP1(Santa Cruz, sc-13939), Dnmt1 (abcam,
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ab13537), Dnmt3a (abcam, ab13888), Dnmt3b (abcam, ab2851), c-Myc
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(Santa Cruz, sc-764), cyclin D1 (Santa Cruz, sc-753), β-catenin (Santa Cruz,
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sc-7199), E-cadherin (Bioworld, BS1098), fibronectin (Santa Cruz, sc-9068)
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and twist (Santa Cruz, sc-15393). Secondary antibodies coupled to HRP were
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purchased from Abcam (ab6789). Proteins of interest were detected with
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Super Signal West Pico Chemiluminescent substrate Kits (Pierce, IL, USA).
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Promoter plasmid constructs
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The serially truncated SFRP1 promoter fragments, with their 5’-ends ranging
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from -2030 to -407 and their 3’-end being fixed at +1, were prepared by PCR
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amplification of human genomic DNA using sense primers containing a KpnI
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restriction site, and all of the constructs shared the same antisense primer
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containing a HindIII restriction site (Supplementary Table 1). The PCR
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products were cloned into pGL3-Basic vector (Promega) and verified by DNA
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sequencing.
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Dural-luciferase assay
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Cells were plated in 24-well plates and transfected with 0.5 μg of each deletion
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construct of SFRP1 promoter (-2030/+1, -1619/+1, -1202/+1, -837/+1 and
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-407/+1) along with 100 ng/well pRL-TK (an internal control) by using
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LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA). At 16 h after
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transfection, cells were infected with AdCore or AdGFP. At 24 h after infection,
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cells were lysed and subjected to dual-luciferase reporter assay (Promega,
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Madison, WI, USA) following the manufacturer’s protocol. Assays were
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performed in triplicate and expressed as means ± S.D.
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Immunoprecipitation
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Huh7 cells were infected with AdCore or AdGFP control. At 24 h after infection,
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whole-cell extracts were collected and incubated with anti-HDAC1 antibody or
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anti-Dnmt1 antibody overnight at 4 °C followed by 2 h incubation with protein G
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agarose beads (Millipore, MA, USA). HDAC1 or Dnmt1 was
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immunoprecipitated, and the immunocomplex was washed (three times) with
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RIPA buffer. Samples were then resolved by SDS/PAGE and subjected to
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western blot assay using anti-core antibody (abcam ab2740).
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Colony formation assay
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Stable HCC cell lines expressing HCV core (designated as SK-Hep1-Core/
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Huh7-Core) or parental cell lines were counted and seeded in 6-well plates
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with a density of 100 cells per plate. Cells were incubated at 37°C for 3 weeks
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with growth media replaced every two days. Colonies were stained with 0.5%
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crystal violet for 25 min and photographed.
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Cell proliferation assay
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Cell proliferation of HCC lines stably transduced with HCV core protein was
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examined by BrdU and MTS incorporation assay. For 5-Bromo
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-2’-Deoxyuridine (BrdU) incorporation assay, Huh7-Core cells or parental cells
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were infected with AdSFRP1, AdsiDnmt1 or AdSFRP1 plus AdsiDnmt1
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respectively. At 24 h after infection, cells were incubated with 10μmol/L BrdU.
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After fixing the cells were reacted with anti-BrdU-peroxidase for 2 h at room
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temperature, and the color developed after adding trimethyl benzidine was
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measured at 490nm. For MTS, SK-Hep1 cells were plated in 96-well microtitre
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plates at a density of 4×103 cells/well and measured at the indicated time
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points post-plated. Cell proliferation was assessed by adding 20 µl of MTS
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labeling reagent into each well and incubating at 37°C for 2 h. The plates were
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read on a microplate reader (Synergy HT Multi-mode Microplate Reader,
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Bio-Tek) at a wavelength of 490 nm.
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Crystal violet cell viability assay
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The crystal violet staining procedure was carried out as described.11 Briefly,
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cells were fixed in 10 % buffered formalin for 20 min and then stained with
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0.5% crystal violet solution at room temperature for 30 min. The plates were
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washed with ddH2O and dried in the air, and ultimately incubated with 33%
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acetic acid to dissolve the dye. Cell viabilities were quantified by measuring the
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absorbance at 570 nm in a microplate reader (Synergy HT Multi-mode
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Microplate Reader, Bio-Tek).
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Cell migration and invasion assay
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In vitro invasion assays were performed using 24-well Transwell unit with
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polycarbonate filters (Corning Costar, Cambridge, MA). Cells infected with
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AdSFRP1, AdsiDnmt1 or AdSFRP1 plus AdsiDnmt1 were suspended at
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density of 5×105/ml in culture medium without FBS and then placed in the
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upper part of the Transwell. Meanwhile, migration-inducing medium (with 10%
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FBS) were added to the lower wells of the chambers. Cells were incubated for
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22 h, fixed with ethanol and stained with 0.05% crystal violet for 30 min. Cells
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in the upper chamber were removed with a cotton swab. Cells that invaded
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through the Matrigel (Matrigel™ Basement Membrane Matirx, BD Biosciences,
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USA) to the underside of the filter (5 fields/filter) were counted. Three invasion
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chambers were used per treated group. The values obtained were calculated
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by averaging the total number of cells from three filters. The experimental
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procedures for in vitro migration assays were the same as the in vitro invasion
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assay described above except that the filters were not coated with Matrigel. All
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experiments were performed in triplicates.
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Tumorigenicity assays in nude mice
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The care and use of experimental animals was in compliance with the
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institutional guidelines approved for our study. Athymic nude mice (4-6 week
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old, male, 18-25g) were used for the studies. Huh7-Core or control stable cells
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were infected with AdsiDnmt1, AdSFRP1, or AdsiDnmt1 plus AdSFRP1 for 15
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h, and collected for subcutaneous injection (1x106/injection) into the flanks of
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athymic nude (nu/nu) mice. Four nude mice were included each group and
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tumor growth was examined every seven days over a course of 8 weeks.
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Tumor volume (V) was monitored by measuring the length (L) and width (W) of
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the tumor with calipers and was calculated with the formula
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V[cm3]=(length[cm)×(width[cm]×(width[cm])/2. At 8 wk after implantation,
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animals were sacrificed, and tumor masses were retrieved for histological
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analysis.
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Dnmt1 and HDAC activity assay
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Nuclear extracts from xenograft samples were isolated using the EpiQuik
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Nuclear Extraction Kit (OP-0002, Epigentek). Equal amounts of nuclear extract
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(5 μg) were applied for Dnmt1 or HDAC activity assay (P-336A and P-4002,
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Epigentek) according to the manufacturer's protocol. Colorimetric assay were
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performed by measuring the absorbance at 450 nm in a microplate reader
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(Synergy HT Multi-mode Microplate Reader, Bio-Tek). Dnmt1 and HDAC
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activity (% of control) were calculated by dividing the sample’s net OD with the
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vector control’s net OD.
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Immunohistochemical staining
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Retrieved xenograft samples were fixed with 4% paraformaldehyde,
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embedded and sectioned. Sections were incubated with β-catenin (Santa Cruz,
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sc-7199), c-Myc (Santa Cruz, sc-764), SFRP1 (Santa Cruz, sc-13939), PCNA
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(Maixin-Bio, MAB-0145, China), MMP-2 (ZSGB-BIO, ZM-0330, China), MMP-9
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(ZSGB-BIO, ZA-0336, China), VEGF (ZSGB-BIO, ZA-0580, China) or Dnmt1
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(abcam, ab13537) antibodies. Subsequently, the slides were incubated with
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EnVision System-HRP (Maixin-Bio, Fuzhou, China) and visualized using DAB
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substrate (Maixin-Bio, Fuzhou, China).
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2. Supplementary figure legends
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Suppl. Figure 1 Validation of HCV replicon and hepatoma cells stably
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expressing HCV core. (A) Semi-quantitative RT-PCR analysis of the mRNA
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expression for SFRP1 to SFRP5 genes after exogenously expressing HCV
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core in Huh7 cells. Cells were treated as described in Fig. 1 A and the total
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RNA was subjected to RT-PCR assay. Experiments were performed in
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triplicate, and representative ones are shown. GAPDH was used as a loading
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control. * Primer dimers. (B) Infection of permissive Huh7.5.1 cells with HCV.
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Huh7.5.1 cells were initially transfected with JFH-1 RNA and the parental
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Huh7.5.1 cells were used as mock control. The expression of the viral proteins
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core, E1 and NS3 were analyzed by western blotting. Recombinant HCV core
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protein was used as positive control. (C) Validation of Huh7 cells or SK-Hep1
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cells stably expressing HCV core. Huh7 or SK-Hep1 cells were infected with
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retroviral virus vector pSEB-3Flag expressing HCV core, and then selected
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with Blasticidin for 3 weeks. Stable cell clones were designated as Huh7-Core
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or SK-Hep1-Core cells. Whole cell lysates were subjected to western blot
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analysis, using anti-core (Abcam) antibody. β-tubulin was used as loading
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control.
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Suppl. Figure 2 HCV Core decreases SFRP1 promoter activity. (A)
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Mapping the minimal promoter region required for core-mediated suppression
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of SFRP1 expression. LO2 cells were transiently co-transfected with pRL-TK
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and pGL3-Basic control or reporter constructs containing various lengths of the
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5’-flanking region of the SFRP1. At 24 h after transfection, cells were then
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infected with AdCore or AdGFP. Results were obtained as relative luciferase
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activity against the activity of pGL3-Basic. Data were shown as mean ± SD of
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three independent experiments. *P<0.05 (Core vs GFP). (B) HCV core protein
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suppresses SFRP1 promoter activity in a dose-dependent manner. Huh7 and
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LO2 cells were transfected with the reporter construct pGL3-S400 (-407/+1)
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and infected with AdGFP or AdCore, respectively. Promoter activities of
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SFRP1 were measured by dual luciferase reporter gene assays. Data are
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present as means ± SD. *P<0.05 (Core vs GFP in Huh7 cells). #P<0.05 (Core
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vs GFP in LO2 cells).
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Suppl. Figure 3 DNA methylation and histone deacetylation in SFRP1
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promoter region in core-transduced SK-Hep1 cells. (A) Western blot
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analysis of Dnmt1, Dnmt3a and HDAC1 expression in core-expressing Huh7
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cells. Cells were infected with AdCore or AdGFP, and then total cell lysates
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were collected and performed western blot analysis at 48 h after infection.
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β-tubulin was used as loading control. (B) Enhanced expression of Dnmt1 and
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HADC1 in core-transduced Huh7 or SK-Hep1 cells. Huh7 cells or SK-Hep1
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cells were infected with AdCore or AdGFP control for 48 h. Cell lysates were
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collected for western blot using anti-Dnmt1 and anti-HDAC1 antibodies
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(GAPDH as loading control). (C) Overexpression of HCV core induces an
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enriched recruitment of DNA methylation and histone deacetylation in SFRP1
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promoter region. Lysates of SK-Hep1 cells infected with AdCore or AdGFP
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control were performed ChIP analysis as described in Fig.3. AdGFP and IgG
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served as controls. (D) Enriched recruitment of Dnmt1 in SFRP1 promoter was
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partially reversed upon Aza treatment. Similar ChIP assays were performed on
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cell lysates from core-expressing SK-Hep1 cells untreated or treated with Aza.
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Suppl. Figure 4 HCV core protein does not physically interact with Dnmt1
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or HDAC1 in vitro. (A) IP/Western blot analysis of interaction between core
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and endogenous HDAC1 in Huh7 cells expressing core protein or empty vector.
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Protein input lysate is shown on the bottom rows. No physical interaction
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between HCV core and HDAC1 proteins was readily detected in Huh7 cells. (B)
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IP/Western blot analysis of interaction between core and endogenous Dnmt1
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in Huh7 cells.
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Suppl. Figure 5
Knockdown of Dnmt1 or overexpression of SFRP1
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suppressed proliferation and migration of SK-Hep1-Core cells. (A) Colony
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formation assay in SK-Hep1-Core cells. Cells were infected with retroviral virus
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expressing HCV core or vector control and cultured in Blasticidin for 3 weeks.
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Cell colonies were stained with crystal violet. Experiments were performed in
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triplicate, and representative ones are shown. (B) Cell viability assay.
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SK-Hep1-Core cells were treated and assayed as described in Fig. 5B. Vector
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transduced and parental SK-Hep1 cell lines were used as controls. *P<0.05
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(compared with the vector mean values). (C) Cell proliferation curves.
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Core-expressing SK-Hep1 cells were treated as described in Fig. 5B and then
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plated into 96-well plate at 0.5×104/ml. Vector transduced and parental
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SK-Hep1 cell lines were used as controls. Cells were counted every 24 h in
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triplicate. Data are present as mean ± S.D. **P<0.01 (SFRP1 vs vector).
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∆P<0.05; ∆∆P<0.01
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vs vector). (D) Knockdown of Dnmt1 or restoration of SFRP1 reduces
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SK-Hep1-Core cell migration. Cells were treated as described in Fig. 5B and
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subjected to transwell assay. Quantitative evaluation of cell migration activity
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was represented as mean ± SD of 5 randomly selected microscopic fields from
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3 independent wells (**P< 0.01 compared with the vector mean values). (E)
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Matrigel invasion assay in core-expressing SK-Hep1 cells. Cells were treated
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as described in Fig. 5B and then allowed to invade through transwell inserts (8
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μm) coated with Matrigel. Cell invasion ability was evaluated as described in
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Fig. 5D. **P<0.01.
(siDnmt1 vs vector). #P<0.05; ## P<0.01 (siDnmt1+ SFRP1
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Suppl. Figure 6 Knockdown of Dnmt1 or overexpression of SFRP1
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decreased aggressive marker expression in xenograft tissues. (A)
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Expression levels of Dnmt1, SFRP1 and HDAC1 in Huh7-Core cells.
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Huh7-Core cells were treated as described in Fig 6 A, and then cell lysate was
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collected for western blotting analysis using anti-SFRP1, anti-Dnmt1 or
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anti-HDAC1 antibody (GAPDH as loading control). (B) (C) Dnmt1 and HDAC
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activities assay. Dnmt1 and HDAC activities in xenograft tissues were
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examined with DNA Methyltransferase 1 Activity/Inhibition Assay Core Kit and
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HDAC Activity/Inhibition Assay Kit (P-3006A and P-4002, Epigentek, Brooklyn,
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NY). Dnmt1 and HDAC activity (% of control) were calculated by dividing the
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sample’s net OD with the vector control’s net OD. *P<0.05 (compared with the
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vector mean values). (D) Immunohistochemical detection of VEGF (panels a to
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e), MMP-2 (f to j), MMP-9 (k to o) and Dnmt1 (p to t) in xenograft tumor
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samples. Representative images are shown. Magnification, × 400.