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Chemicals
Iodoacetamide, KBr, TEMED, urea, thiourea, ammonium bicarbonate and α-cyano-4hydroxycinnamic acid (CHCA) were from Sigma Aldrich. DTT, TFA and glycerol were from
Fluka. SDS, CHAPS, Trizma base, glycine, 30% acrylamide/Bis solution 29:1 (3,3% C) and Sypro
Ruby were from Bio-Rad. Methanol, acetone, acetic acid, and acetonitrile were acquired from Carlo
Erba Reagenti. Ammonium persulfate (APS) and NaBr were from ICN Biomedicals Inc. Porcine
trypsin was from Promega. IPG buffer pH 3-10 NL, IPGs 3-10 NL and dry strip cover fluid were
purchased from GE Healthcare. Calibration mixture for peptide mass fingerprinting (des-Argbradykinin, angiotensin I, Glu 1-fibrinopeptide B, neurotensin, adrenocorticotropic hormone
(ACTH) clip 1–17, ACTH clip 18–39, and ACTH clip 7–38 with masses: 904.468, 1296.6853,
1570.6774, 1672.9175, 2093.0867, 2465.1989, and 3657.9294 Da, respectively) was purchased
from Applied Biosystems.
Proteins isolation
Proteins from lipoproteins preparation were delipidated and precipitated accordingly to Mastro and
Hall using acetone:methanol (8:1) procedure. Subsequently, the precipitated proteins were boiled in
a solution containing 325 mM DTT, 4% CHAPS, and 45 mM Tris for 5 min, and cooled to room
temperature. In order to remove salts five volumes of ice cold acetone was added to the sample and
incubated 90 min at 4ºC, centrifuged (2800 x g for 15 min at 4ºC) and then air-dried. Proteins were
resuspended in 1-D buffer (6M Urea, 2M Thiourea, 4% CHAPS, 1% DTT, 2% v/v IPG Buffer (47). Proteins concentration was determined with 2-D Quant Kit (GE Healthcare).
Two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis was performed using IPGphor II (GE Healthcare). Proteins
(300μg) were loaded on a pH 3-10 NL IPG strips (IPGs) by in-gel rehydratation for 9h, using low
voltage (30V). The proteins were then electrofocused at 70000 Vh at maximum voltage of 8000 V.
Before second dimension (SDS-PAGE) IPGs were subject to protein reduction and alkylation by
two sequential immersion in equilibration buffer (6 M urea, 2% SDS, 30% glycerol, and 50 mM
TrisHCl pH 8.8) containing 1% DTT for 10 min, and in the equilibration buffer with 4%
iodoacetamide for 10 min. At the end of this alkylation treatment IPG strips were loaded onto
12%T-3.3%C separating gels (1 + 18 + 18 mm) and sealed in place with an agarose overlay (0.5%
low melting agarose, 0.1% SDS, 192 mM glycine and 25 mM Tris pH 8.8). The second dimension
was performed at 50 mA costant for about 6 h. Proteins were revealed with Sypro Ruby staining
and visualized using a Typhoon 9200 laser scanner (GE Healthcare).
Image analysis
The 2-DE experiment was repeated twice for each sample and quantitative differences were
analyzed using the ImageMaster™ 2D Platinum 6.0 software (GE Healthcare). Briefly, protein
spots were automatically detected and then refined manually; subsequently the gels were
normalized, and matched. For each spot, the ratio between its volume and the sum of all the spots
volume in the gel were calculated and used for quantitative comparison. The differences among
protein spots were considered significant when similar quantitative changes were found in all paired
gels and when the normalized volume spots ratios were at least two-fold and the p-value ≤0.05.
Protein excision and tryptic digestion
Protein spots were excised from the gel using InvestigatorTM ProPic spot picker (Genomic
SolutionsTM) and transferred to small Eppendorf tubes (0.2 mL). Protein-containing gel pieces were
destained with 50μl of 0.1 M ammonium bicarbonate (5 min at RT). Then, 50 μl of 100%
acetonitrile were added to each tube and incubated for 5 min at RT. The liquid was discarded, the
washing step repeated once and the gel plugs were shrunk by addition of acetonitrile. The dried gel
pieces were re-swollen with 4.5 ng/μl trypsin in 50 mM ammonium bicarbonate and digested
overnight at 37°C. Peptides were concentrated with ZipTipμC18 pipette tips (Millipore). Co-elution
was performed directly onto a MALDI target with 1 μl of α-cyano-4-hydroxycinnamic acid matrix
(5 mg/ml in 50% acetonitrile, 0.1% TFA).
Protein Identification by Mass Spectrometry
MALDI-MS and MALDI-MS/MS were performed on an Applied Biosystems 4700 Proteomics
Analyzer with TOF/TOF ion optics. Data were acquired in positive MS reflector mode with five
spots of standard (ABI4700 Calibration Mixture) for calibration. Mass spectra were obtained from
each sample spot by 30 sub-spectra accumulation (each 7 consisting of 50 laser shots) in a 750 to
4,000 mass range. Five signal-to-noise best peaks of each spectrum were selected for MS/MS
analysis. For MS/MS spectra, the collision energy was 1 keV and the collision gas was air. The
interpretation of both the MS and MS/MS data was carried out by using the GPS Explorer software
(Version 1.1, Applied Biosystems), which acts as an interface between the Oracle database
containing raw spectra and a local copy of the MASCOT search engine (Version 1.8). Peptide mass
fingerprints obtained from MS analysis were used for protein identification in the NCBI nonredundant database. All peptide mass values are considered monoisotopic and mass tolerance was
set at 50 ppm. Trypsin was given as the digestion enzyme, 1 missed cleavage site was allowed,
cysteine carboamidomethylated was assumed as fixed modification, methionine was assumed to be
partially oxidized and serine, threonine and tyrosine partially phosphorylated. Mascot (Matrix
Science) scores greater than 61 were considered significant (p<0.005). For MS/MS analysis, all
peaks with a signal-to-noise ratio greater than 5 were searched against the NCBI database using the
same modifications as the MS database, with a fragment tolerance less than 0.3 Da.
Densitometric analysis
Proteins associated to the LDL isolated from single donors were analyzed on precasted SDSpolyacrylamide gradient gels (3-8% and 4-12%; Invitrogen). Protein bands were stained with Sypro
Ruby and detected by Typhoon 9200 laser scanner (GE Healthcare). Densitometric quantification of
protein bands corresponding to apoE, apoA-I and apoB-100 were performed using ImageQuant V
5.0 software (GE Healthcare). Protein bands identity was verified by mass spectrometry analysis
(data not shown).
Western Blotting and ELISA assay
Western blotting was carried out as previously described[28]. For western blot analysis of apoB-100
precasted SDS-polyacrylamide gradient gels (3-8%; Invitrogen) were used.
Antibodies used in the present study include mouse anti-apoA-I (human-specific) and anti-tubulin
(SC-58230 and SC-32293, Santa Cruz Biotechnology), mouse anti-apoB and anti-apoE (611400
and 610449, BD Transduction Laboratories), rabbit anti-NS5A (Ab2594, Abcam) and mouse antiNS3 (MO-I40018Q Anogen).
ELISA Kits specific for apoA-I (3710-1HP) and apoB (3715-1HP) were purchased from Mabtech
and used accordingly to the manufacturer’s instructions. For ELISA assays LDL produced in
culture were concentrated by vivaspin (Sartorius Stedim Biotech).
The human specificity of the anti-apoA-I antibodies used for western blotting and ELISA assay
were tested using either the culture medium or the lipoproteins isolated from the culture medium as
control.
Immunoprecipitation
Immunoprecipitation of apoB was performed on low density particles released in the medium by
Huh7 and RepBlast cells. Lipoproteins were isolated as described in material and method section.
Lipoproteins were concentrated and washed with 1XPBS at 3000 rpm at 5°C for 3h using vivaspin
columns (Sartorius Stedim Biotech). Equal amount of lipoproteins were brought to a final volume
of 1 ml with IP buffer (100 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA) containing protease
inhibitor mixture and subjected to overnight immunoprecipitation using a goat anti- apoB-100
antibody (AB742 Chemicon International). The next day, 25µl of protein G-Sepharose beads
(Amersham Biosciences) blocked with 4% BSA were added and incubated with the samples for 2h
at 4°C. The protein-beads complexes were pelleted by centrifugation for 3 minutes at 1500 rpm.
Immunoprecipitates were washed five times with IP wash buffer (100 mM Tris-HCl pH 8, 100 mM
NaCl) and solubilized by boiling in SDS sample buffer. Immunoprecipitates flow-through were
collected and concentrated again to a final volume of 100 l. Proteins were resolved by 3-8%
(NuPage, Invitrogen) or 12% SDS-PAGE, transferred to nitrocellulose membranes, and visualized
by immunoblotting with mouse anti-apoB (BD Biosciences) and mouse anti-apoA-I (Santa Cruz
Biotechnologies, USA) antibodies.
RNA interference
ApoA-I RNA interference was performed using oligonucleotides from Invitrogen (IDs HSS166915,
HSS166916, HSS166917). All the experiments showed in the manuscript were performed with the
oligo HSS166917. A scrambled siRNA containing a similar GC content was used as transfection
control. 1.5x105 cells/well were transfected with 200 pmol siRNA in 6 well plates by Lipofectamine
RNAiMAX (Invitrogen) as indicated by the supplier.
For apoA-I silencing in the Rep60, RepBlast and NNeo/C-5B cell lines transfection was repeated on
two consecutive days to increase transfection efficiency. RNA decrease was checked by real time
PCR and western blot analysis 3 and 6 days after transfection.
For apoA-I silencing in the Huh7 cells transfection was done once and the day after cells were
infected with Huh7.5.1-generated HCVcc at MOI=0,1. RNA decrease was checked by real time
PCR and western blot analysis 3 and 6 days after transfection.
Real Time PCR
Cellular RNA was prepared with Trizol reagent (Invitrogen), while viral RNA present in the
supernatants was prepared with a purification kit (Quiagen), accordingly to the manufacturer’s
instructions. cDNA synthesis was generated from 1 g of RNA using the reverse transcription kit
(Promega) according to manufacturer’s recommendations. Real time PCR reactions were performed
with the LightCycler (Roche) using the LightCycler FastStart DNA Master SYBR-Green-I (Roche)
according to the manufacturer’s instructions. 5 l of 1:10 cDNA dilution cDNA was used as
template and cycling parameters were 95°C for 10 min, followed by 40 cycles of 95°C for 10s,
62°C for 10s, 72°C for 10s. Relative amounts were obtained using the 2–ΔΔCt method normalized for
the L34 gene expression. Primer sets for all amplicons were:
ApoA-I forward: 5’-CAAGATGAACCCCCCCAGAG-3’
ApoA-I reverse: 5’-CTGTTTTCCCAAGGCGGAG-3’
Subgenomic Replicon forward: 5’-TACTCCCAACAGACGCGAGG-3’
Subgenomic Replicon reverse: 5’-GCAGGTCGCCAGGAAAGATT-3’
Genomic Replicon forward: 5’-AGCCAGACAGGCTATAAAATCGC-3’
Genomic Replicon reverse: 5’-CCGCAGCTAGTCGTCAGCAC-3’
HCVcc forward: 5’-TCTGCGGAACCGGTGAGTA-3’
HCVcc reverse: 5’-TCAGGCAGTACCACAAGGC-3’
L34 forward: 5’-GTCCCGAACCCCTGGTAATAG-3’
L34 reverse: 5’-GGCCCTGCTGACATGTTTCTT-3’
HCV generated in the culture medium: HCV RNA copy number quantification and
infectivity.
Huh7 cells were transfected with either CTR-L or apoA-I siRNA as described above, cultured for
24 hours, infected with HCV at MOI=0.1. After 2 hours cells were washed 2 times with 1XPBS and
then cultured for further 4 days. RNA present in both culture medium and intracellularly was
collected daily and isolated as described above. Quantitation was carried out using an external
standard curve constructed by amplifying known amounts of a vector coding for the HCV JFH-1
genome (serial 10-fold dilutions ranged from 7,24x10-1 to 7,24x107 copy numbers). Triplicates of
cycle numbers (CT) were plotted against the logarithmic copy numbers. The PCR amplification
efficiency was controlled by the slope of the standard curve (Slope =3.225; E= 104.2%). RNA copy
numbers were calculated by interpolation of Ct values generated in the standard regression curve.
All the data were then calculated as relatively to the CTR-L siRNA-treated cells at day 1 postinfection.
The infectivity of HCV particles generated in culture, by both CTR-L and apoA-I siRNA-treated
cells at day 4 post-infection, was tested by infecting naive Huh7.5.1 cells. After 48 hours of culture
the intracellular HCV RNA levels were isolated and quantified by RT-qPCR as described above.