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Transcript
1
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
5
The HCV core protein coding region (genotype 1a) was PCR-amplified and
6
cloned into a retroviral vector pSEB-3Flag that also conferred resistance to
7
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
25
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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28
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,
60
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
84
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%
109
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
117
assay described above except that the filters were not coated with Matrigel. All
118
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
123
old, male, 18-25g) were used for the studies. Huh7-Core or control stable cells
124
were infected with AdsiDnmt1, AdSFRP1, or AdsiDnmt1 plus AdSFRP1 for 15
125
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
130
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
132
analysis.
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Dnmt1 and HDAC activity assay
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Nuclear extracts from xenograft samples were isolated using the EpiQuik
136
Nuclear Extraction Kit (OP-0002, Epigentek). Equal amounts of nuclear extract
137
(5 μg) were applied for Dnmt1 or HDAC activity assay (P-336A and P-4002,
138
Epigentek) according to the manufacturer's protocol. Colorimetric assay were
139
performed by measuring the absorbance at 450 nm in a microplate reader
140
(Synergy HT Multi-mode Microplate Reader, Bio-Tek). Dnmt1 and HDAC
141
activity (% of control) were calculated by dividing the sample’s net OD with the
142
vector control’s net OD.
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Immunohistochemical staining
145
Retrieved xenograft samples were fixed with 4% paraformaldehyde,
146
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
170
analysis, using anti-core (Abcam) antibody. β-tubulin was used as loading
171
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
196
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
201
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
218
triplicate, and representative ones are shown. (B) Cell viability assay.
219
SK-Hep1-Core cells were treated and assayed as described in Fig. 5B. Vector
220
transduced and parental SK-Hep1 cell lines were used as controls. *P<0.05
221
(compared with the vector mean values). (C) Cell proliferation curves.
222
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
225
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
241
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
243
activities assay. Dnmt1 and HDAC activities in xenograft tissues were
244
examined with DNA Methyltransferase 1 Activity/Inhibition Assay Core Kit and
245
HDAC Activity/Inhibition Assay Kit (P-3006A and P-4002, Epigentek, Brooklyn,
246
NY). Dnmt1 and HDAC activity (% of control) were calculated by dividing the
247
sample’s net OD with the vector control’s net OD. *P<0.05 (compared with the
248
vector mean values). (D) Immunohistochemical detection of VEGF (panels a to
249
e), MMP-2 (f to j), MMP-9 (k to o) and Dnmt1 (p to t) in xenograft tumor
250
samples. Representative images are shown. Magnification, × 400.