Supporting Methods
Analysis of Transcription Factor Binding Sites. Using the UCSC Genome Bioinformatics site, we
downloaded the TSS for all human RefSeq genes, defined by the July 2009 refGene table (1). The region
2kb around the TSS for all human genes were identified, and sequences were obtained for mouse
(mm9), human (hg18) and chimpanzee (panTro2) as acquired through the multiple genome alignment,
multiz30way (2), based off of the human genome also available through the UCSC Genome
Bioinformatics Site.
Transcription factor binding sites were either obtained from the TRANSFAC
database (3) (all high-quality vertebrate transcription factor matrices for v. 2008.3) or independent
publications (4). The MATCH algorithm (5) was used to scan the human sequences and aligned
sequences for putative binding sites using TRANSFAC defined cutoff scores to minimize the sum of false
positive and false negative matches. Putative binding sites were considered to be evolutionarily
conserved if a match occurred in the same position for all aligned species of interest and no gap was
present in the alignment of any of those species. Each TRANSFAC matrix was linked to a set of gene
symbols describing the potential binding factors using annotations present in the “Binding Factor” field
of the matrix. Only vertebrate TRANSFAC matrices that could be linked to a HGNC gene symbol (Dec.
2008) (6), either directly or through an alias listed in NCBI gene were included.
RNA isolation, reverse transcription and qPCR. Total RNA was harvested and extracted from cytokine or
inhibitor-treated Huh7 cells and human hepatocytes using RNeasy Mini Kit (Qiagen) according to the
manufacturer’s instructions. RNA was converted into cDNA using High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems). Quantitative PCR was performed using 7500 Real Time PCR
System (Applied Biosystems). Each reaction is composed of cDNA reverse transcribed from 100 ng of
total RNA, 12.5 μl of Power SYBR Green PCR master mixture (Applied Biosystems), and 200 nM forward
and reverse primers in a total volume of 25 μl. QPCR was performed using the following program: stage
one with an initial incubation at 50 °C for 2 min, stage two with incubation at 95 °C for 10 min, and stage
three consisting of 30 s at 95 °C followed by 1 min at 60 °C for 50 cycles. Gene expression was analyzed
using ABI SDS v1.2.3 software (Applied Biosystems) with normalization to GAPDH expression level. The
primer sequences used were as follows:
MX1, 5'-GTG GCT GAG AAC AAC CTG TG-3' (forward)
5'-GGC ATC TGG TCA CGA TCC C-3' (reverse)
IRF9, 5'-GAT ACA GCT AAG ACC ATG TTC CG-3' (forward)
5'-TGA TAC ACC TTG TAG GGC TCA-3' (reverse)
IFIT3, 5'-TCA GAA GTC TAG TCA CTT GGG G-3' (forward)
5'-ACA CCT TCG CCC TTT CAT TTC-3' (reverse)
CXCL10, 5'-GTG GCA TTC AAG GAG TAC CTC-3' (forward)
5'-TGA TGG CCT TCG ATT CTG GAT T-3' (reverse)
RSAD2, 5'-CAA GAC CGG GGA GAA TAC CTG-3' (forward)
5'-AAC TCT ACT TTG CAG AAC CTC AC-3' (reverse)
TNFSF10, 5'-TGC GTG CTG ATC GTG ATC TTC-3' (forward)
5'-GCT CGT TGG TAA AGT ACA CGT A-3' (reverse)
GAPDH, 5'-CAT GAG AAG TAT GAC AAC AGC CT-3' (forward)
5'-AGT CCT TCC ACG ATA CCA AAG T-3' (reverse)
The specificity of primers was verified by dissociation analysis using 7500 Real Time PCR system and the
expected amplicon size of amplified products was visualized on 1.5% agarose gels.
Immunoblot analysis. Cytokine or inhibitor-treated Huh7 cells were washed with ice-cold phosphatebuffered saline before lysis with RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0). Cells were scraped off from the 6-well plates and agitated
at 4 °C for 10 min. After centrifugation at 12,000 rpm at 4 °C for 20 min, supernatants were mixed at a
1:1 ratio with Laemmli 2X buffer. Protein lysates were heated at 95 °C for 5 min, separated by 10% MiniPROTEAN®TGXTM gels (BIO-RAD), transferred onto 0.45 μm nitrocellulose membranes, and probed with
antibodies specific for p-STAT1 (Y701), p-STAT1 (S727), STAT1, (Cell Signaling), and β-actin (Santa Cruz
Biotechnology). Secondary incubation was performed using anti-rabbit IgG HRP-linked antibody (Cell
Signaling) and rabbit anti-goat HRP-conjugated antibody (Santa Cruz Biotechnology). Proteins were
visualized using HyGLO chemiluminescent HRP antibody detection reagent (Denville Scientific Inc.) and
Fuji LAS-3000 cooled CCD camera.
Inhibitor treatment. Huh7 cells were treated with the following kinase inhibitors at 37°C for 60 min
before stimulation with 500 U/ml of either IFN-α or IFN-β. The concentrations of inhibitors were used as
follows: the p38 MAPK inhibitor SB203580 at 10 μM; the PKC-δ-specific inhibitor rottlerin at 2 μM; the
MEK inhibitor PD98059 at 30 μM; the PI3K inhibitor LY294002 at 20 μM; the JNK inhibitor SP600125 at
25 μM, and the protein translation inhibitor cycloheximide at 100 μg/ml. Stock solutions were prepared
in dimethyl sulfoxide (DMSO) at approximately 1,000× concentration with respect to the working
concentrations indicated above. DMSO alone was added to Huh7 cells in all experiments to serve as the
vehicle control.
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