Supplementary Data - Journal of Hepatology

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Supplementary Data
Supplementary Materials and methods
Materials
The antibodies used were: Negative control IgG (Active Motif), HNF-1α, LXRα/β, βactin and PARP1/2 (Santa-Cruz), Mirk/Dyrk1B (Cell Signaling), HCV core mAb for
immunofluorescence analysis (Alexis Biochemicals) and HCV NS5A (Biodesign
International). The polyclonal antibodies for HCV core and core+1 have been
published elsewhere [1S,14]. The human HNF-1α expression plasmid and the (929/+109) human ANGPTL-3 promoter construct were kind gifts from Prof.
Hadzopoulou-Cladaras (Aristotle University of Thessaloniki, Greece) and Dr Delerive
(GSK Research Laboratories), respectively. Huh7-Lunet and Huh7.5 cells were
supplied from Dr R. Bartenschlager (University of Heidelberg, Germany).
Plasmids
Mutations on the (-89/-74) HNF-1α and (-114/-99) LXR cis-acting elements of the
ANGPTL-3 promoter were carried out by standard site-directed mutagenesis, so that
the sites were abolished without altering local promoter topology, according to
transcription factor prediction analysis (see Suppl. Table 2). Construction of all
expression plasmids coding for HCV proteins used in this study is outlined in
Supplementary Table 1. All constructs were subjected to sequencing analysis.
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Cells, Viruses and viral RNA analysis
Maintenance of the human hepatoma HepG2, Huh7.5 and Huh7-Lunet cell lines has
been outlined in [2S,3S,4S]. The construction of HCV-1a core-expressing cell line
(C2-3) and empty vector control (pTRE), has been described elsewhere [2S].
Viral infections of Huh7.5 cells with the JFH-1 infectious clone were carried out
essentially as described in [3S]. In vitro transcription of the pFK-JFH1wt full length
construct and electroporation of Huh7-Lunet cells has been described elsewhere [4S].
All infections were carried out at MOI (multiplicity of infection) =1.
mRNA Expression analysis
Total RNA was isolated from human liver biopsies and cells using the RNeasy™
Micro and Mini kits (Qiagen), respectively. RT-PCR was carried out using 0.2-1µg
RNA. For semi-quantitative PCR, all conditions were in the exponential phase of
amplification providing direct correlation between the amount of products and RNA
template abundance in the samples. PCR products were analysed on a 2%(w/v)
agarose gel. Quantity-One software (BioRad) was employed for densitometric
analysis and values were normalised to 28S rRNA to offer relative mRNA expression.
For quantitative PCR, Maxima® SYBR green qPCR mix (Fermentas) and the Corbett
Rotor Gene 6000 PCR machine were used. Results were analysed with the internal
standard-curve method and normalised to 18S rRNA. All experiments were
performed in triplicate and histograms were used for representation as described in the
transfection analysis section. The specific gene primers used are shown in
Supplementary Table 2.
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Protein Analysis
Cellular fractionation was performed with the ProteoJet™ Cytoplasmic and Nuclear
Extraction kit (Fermentas). All fractions were analysed for fractionation efficiency by
β-actin (cytoplasm) and PARP1/2 (nucleus) Western blotting. Whole cell extract
preparation, total protein determination and Western blot analysis have been
described elsewhere [5S]. Quantity-One software was used for densitometric analysis
of bands and histograms were prepared as described in the transfection analysis
section. ANGPTL-3 protein levels were determined using the ANGPTL-3 ELISA kit
(Adipogen) according to the manufacturer’s instructions.
For co-immunoprecipitation, whole cell extracts (500μg) were pre-cleared (1.5h, 4ºC)
with Protein A/G agarose beads (Santa-Cruz). The pre-cleared supernatant was
incubated overnight at 4ºC with 3μg/ml anti-HNF-1α antibody. The protein-antibody
complex was captured on Protein A/G agarose beads at 4ºC for 3h. The beads were
collected by centrifugation (14,000rpm, 5min) and after three washes with detergentfree whole cell extract buffer, they were separated on SDS-PAGE and analysed by
Western blot.
Chromatin Immunoprecipitation Assays (ChIP)
ChIP analysis was performed using the ChIP-IT Express kit (Active Motif)
according to the manufacturer’s instructions. The immunoprecipitated DNA fractions
and input control were subjected to qPCR with primers spanning both the HNF-1α
and LXR binding site of the ANGPTL-3 promoter, as suggested by the manufacturer
(Suppl. Table 2).
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Immunofluorescence Analysis
Immunofluorescence and confocal microscopy were carried out as previously
described [6S]. All experiments were performed in triplicate.
Electrophoretic mobility shift assays (EMSA)
EMSA was carried out essentially as described in [5S]. The radiolabelled
oligonucleotides used spanned both the HNF-1α and LXR binding sites of ANGPTL3 promoter (Supplementary Table 2).
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Supplementary Table 1. Information on construction of expression plasmids
used in this study.
Name
Protein
Coding Region Method of cloning
pHPI1320
(c191)
pHPI1800
(c173)
pHPI1802
(c120)
pHPI8130
HCV-1a
core
HCV-1a
core
HCV-1a
core
HCV-1a
core
Full length
(1-191aa)
1-173aa
pHPI8198
HCV-3a
core
HCV-3a
core
HCV-1b
(Met91)
core
HCV-1b
(Met91)
core
pHPI8184
pHPI8167
pHPI8164
pHPI1579
pHPI 1602
HCV-1a
core+1/S
HCV-1a
NS3NS5B
Cloned
Vector
NdeI fragment from SmaI /pCI
pHPI1319
PCR from pHPI1320 SmaI /pCI
1-120aa - His NdeI-BstYI fragment
tag
from pHPI8130
1-120aa
NdeI-BstYI fragment
from pET 20b(+)
(prokaryotic
expression)
Full length
NheI-XbaI fragment
(1-191aa)
from pHPI8184
Full length
PCR
from
3T2
(1-191aa)
(PUC19)
Full length
NheI-XbaI fragment
(1-191aa)
from pHPI8164
Full length
(1-191aa)
PCR from pHPI1589
in/ REF
[7S]
SmaI /pCI
NheI-XbaI
/pCI
[8S]
NheI-XbaI
/pCI
[9S]
[10S]
[11S]
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Supplementary Table 2. Sequences of gene-specific primers used in various
analyses.
Primer Name
Primer Sequence (5’3’)
ANGF
GATTTAGCATTGATCTAACTCAATGTGGAAGAAGG
TTACATTCGTGCAA
GAGGAACAATAAAAAGAAGGAGCTT
GTTAACACGGCTATGGATTACTATGTTCACCTACC
GGTAGGTGAACATAGTAATCCATAGCCGTGTTAAC
CTAACTCAATGTGGAAGAGGGCCCCATTCGTGCAA
GTTAACACG
CGTGTTAACTTGCACGAATGGGGCCCTCTTCCACAT
TGAGTTAG
TCTATCGCTGCAAACCAGTG
ANGCR
mHNF-1F
mHNF-1R
mLXRF
mLXRR
ANGPTL-3F
ANGPTL3-R
qANGPTL3F
qANGPTL3R
qHNF-1αF
qHNF-1αR
qNS3-2aF
qNS3-2aR
ANGEF
ANGER
28S rRNAF
28S rRNAR
18S rRNAF
18S rRNAR
Mirk/Dyrk1B F
Mirk/Dyrk1B R
CGGTCTGGAGAAGGTCTTTG
CCAGAACACCCAGAAGTAACT
TCTGTGGGTTCTTGAATACTAGTC
GTACTCAGCAGGCTGTGGCT
TCTCCCCCAGTAAGGTCCAC
CTGCCACCCTGGGGTTTGGG
GCAGCCCCCATCGGCGAGAA
GGAAGAAGGTTACATTCGTGCAAGTTAACACGGCT
TAA
CATAGTTAATCATTAAGCCGTGTTAACTTGCACGA
ATG
TGAACTATGCTTGGGCAGGG
AGCGCCATCCATTTTCAGGG [10S]
CTCAACACGGGAAACCTCAC
CGCTCCACCAACTAAGAACG [11S]
ATGACTACATCGTGCGCAGT
ATAGGCTTTCACCACCTGGC
Type of
Analysis
ChIP
Mutagenesis
Mutagenesis
PCR
qPCR
qPCR
qPCR
EMSA
PCR
qPCR
qPCR
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Supplementary References
[1S] Budkowska A, Kakkanas A, Nerrienet E, Kalinina O, Maillard P, Horm SV, et
al. Synonymous mutations in the core gene are linked to unusual serological profile in
hepatitis C virus infection. PLoS One 2011;6:e15871.
[2S] Katsarou K, Tsitoura P, Georgopoulou U. MEK5/ERK5/mef2: a novel signalling
pathway affected by hepatitis C virus non-enveloped capsid-like particles. Biochim
Biophys Acta 2011;1813:1854-1862.
[3S] Kato T, Date T, Murayama A, Morikawa K, Akazawa D, Wakita T. Cell
culture and infection system for hepatitis C virus. Nat Protoc 2006;1:22334-22339.
[4S] Lohmann V. HCV replicons: Overview and basic protocols. In: Tang H ed.
Hepatitis C: Methods and Protocols Volume 510, 2nd ed. Humana Press 2009:145162.
[5S] Foka P, Irvine SA, Kockar F, Ramji DP. Interleukin-6 represses the transcription
of the CCAAT/enhancer binding protein-alpha gene in hepatoma cells by inhibiting
its ability to autoactivate the proximal promoter region. Nucleic Acids Res
2003;31:6722-6732.
[6S] Katsarou K, Lavdas AA, Tsitoura P, Serti E, Markoulatos P, Mavromara P,et al.
Endocytosis of hepatitis C virus non-enveloped capsid-like particles induces MAPKERK1/2 signaling events. Cell Mol Life Sci 2010;67:2491-2506.
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[7S] Tsitoura P, Georgopoulou U, Pêtres S, Varaklioti A, Karafoulidou A, Vagena D,
et al. Evidence for cellular uptake of recombinant hepatitis C virus non-enveloped
capsid-like particles. FEBS Lett. 2007;581:4049-4057.
[8S] Karamitros T, Kakkanas A, Katsoulidou A, Sypsa V, Dalagiorgou G, Mavromara
P, Hatzakis A. Detection of specific antibodies to HCV-ARF/CORE+1 protein in
patients treated with pegylated interferon plus ribavirin. J Viral Hepat 2012;19:182188.
[9S] Boumlic A, Vassilaki N, Dalagiorgou G, Kochlios E, Kakkanas A,
Georgopoulou U, Markoulatos P, et al. Internal translation initiation stimulates
expression of the ARF/core+1 open reading frame of HCV genotype 1b. Virus Res
2011;155:213-220.
[10S] Kong SE, Hall JC, McCauley RD. Estimation of gene expression within the
intestinal mucosa using semiquantitative reverse transcriptase-polymerase chain
reaction. Anal Biochem 1999;271:111–114.
[11S] Tsitoura E, Thomas J, Cuchet D, Thoinet K, Mavromara P, Epstein AL.
Infection with herpes simplex type 1-based amplicon vectors results in an IRF3/7dependent, TLR-independent activation of the innate antiviral response in primary
human fibroblasts. J Gen Virol 2009;90:2209-2220.
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Supplementary Figure legends
Supplementary Fig. 1. (A) EMSA analysis of factors binding to an oligonucleotide
containing the HNF-1α and LXR sites of the ANGPTL-3 promoter using nuclear
extracts from pTRE/C2-3 cells. The free probe (FP) is shown by a labelled arrow to
verify equal loading. (B1-B2) Overexpression of HNF-1α in pTRE/C2-3 HepG2 cells
does not alleviate core-mediated down-regulation of ANGPTL-3 promoter activity.
Cells were transfected with pANGPTL-3 promoter, pCI (V), HNF-1α and c191 (in
HepG2 cells) expression plasmids. (C1-C2) Densitometric analysis of Western blots
presented in Fig.2E1-E2 in HepG2 (C1) and pTRE/C2-3 (C2) cells. (D1). ANGPTL-3
mRNA levels in Huh7-Lunet cells transfected with JFH-1 full length replicon RNA at
given time points. (D2) NS3 expression was used to monitor replication efficiency.
Supplementary Fig. 2. (A) Comparative amino-acid sequence analysis of core
proteins of genotypes 1a, 1b and 3a. (B) Long-term infection of Huh7.5 cells with the
JFH-1 clone. Isolated RNA was subjected to qRT-PCR with HNF-1α and ANGPTL3-specific primers and compared to mock-infected cells. (d = days).
Supplementary Fig. 3. (A) HepG2 cells co-transfected with pANGPTL-3 promoter
and c191 expression plasmid or vector pCI (V), were subjected to treatment with the
proteasomal inhibitor MG-132 (MG) or vehicle (D) for 8h. (B) Cellular extracts from
the transfection described in (A) were subjected to immunoblotting with anti-HNF-1α
and β-actin antibodies. Densitometric analysis of bands is presented underneath, with
HNF-1α values normalised to β-actin. (C) RNA from Huh7.5 cells infected with the
JFH-1 clone for the indicated time points was subjected to qRT-PCR with
Mirk/Dyrk1B-specific primers.

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