Supplemental Materials and Methods
Protein Extraction from HSCs. Quiescent and activated HSCs were
stringently washed with cold PBS and lysed at 4°C with M-PER (Mammalian
Protein Extraction Reagent, Pierce, Rockford, IL) (107cells/ml). The lysates
were centrifuged at 15000×g for 20 min at 4°C, and the supernatants were
collected. Protein concentration was measured by bicinchoninic acid (BCA)
protein assay (Pierce).
iTRAQ Labeling, Sample Cleaning and Desalting. All the reagents and
buffers needed for iTRAQ labeling were supplied by Applied Biosystems
(Applied Biosystems, Foster City, CA), and iTRAQ labeling was performed
according to manufacturer’s instructions. Briefly, 100μg total cell lysates from
three independent experiments were precipitated with ice-cold acetone
overnight at -20°C. After centrifugation, the pellets were dissolved,
denaturated, alkylated and digested with trypsin (Promega, Madison, WI), then
the digestions from quiescent and activated HSCs were labeled with 114 and
116 iTRAQ reagents respectively. Having incubated at room temperature for 1
h, the two samples were pooled.
Before online 2D nano-LC/MS/MS analysis, iTRAQ labeled samples were
cleaned up and desalted. Cation exchange cartridge system (Applied
Biosystems) was employed to remove the reducing reagent, SDS, excess
iTRAQ reagents, undigested proteins and trypsin in the labeled samples.
Afterwards, the elution of cation exchange was diluted and desalted by using
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Agilent HPLC and a 4.6mm id×150mm C18 RP column (Agilent, Waldbronn,
Germany). Elution was dried in a Heto vacuum concentrator (Heto-Holten A/S,
Allerod, Denmark).
Online 2D nano-LC-MS/MS. Two dimensional nanoscale liquid
chromatography and tandem mass (2D nano-LC-MS/MS) analyses were
conducted on a nano HPLC system (Agilent) coupled to a hybrid Quadrupole
Time-Of-Flight (Q-TOF) mass spectrometer (QSTAR XL, Applied Biosystems)
equipped with a nano-electrospray ionization (ESI) source (Applied
Biosystems) and a nano-ESI needle (Picotip, FS360-50-20, New objective Inc.,
Woburn, MA). Analyst™ 1.1 software was used to control QSTAR XL mass
spectrometry, nano HPLC system and acquire mass spectra. Vacuum dried
iTRAQ labeled peptides were reconstituted in phase A and injected at a flow
rate of 10 μl/min onto a high resolution strong cation exchange (SCX) column
(Bio-SCX, 300μm id×35mm, Agilent), which was on-line with a C18 pre-column
(PepMap 300μm id×5mm, Dionex LC Packings, Idstein, Germany). After
loading, the SCX column and C18 pre-column were flushed with 16 step
gradient sodium chloride solution (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,
125, 150, 200, 300 and 400 mM) for 5 min and phase A for 10 min at a flow
rate of 10μl/min. Afterwards, the pre-column was switched on-line with a
nano-flow RP column (Magic C18 , 100μm id×150mm, Michrom Bio
Resources Inc, Auburn, CA), peptides concentrated and desalted on
pre-column were separated using a 120 min linear gradient, from 12% to 30%
phase B (0.1% FA in ACN), at a flow rate of 350nl/min.
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The Q-TOF was operated in the positive-ion mode; ion spray voltage (IS)
typically maintained was 1.8kV. Mass spectra of iTRAQ labeled samples were
acquired in an information-dependent acquisition (IDA) mode, the analytical
cycle consisted of a 0.7s MS survey scan (400-1600m/z) followed by three 2s
MS/MS scans (100-2000m/z) of the three most abundant peaks (i.e. precursor
ions) which were selected from MS survey scan. Precursor ions selection was
based upon ion intensity (peptide signal intensity above 25 CPS (counts per
second)) and charge state (+2-+5), once the ions were fragmented in MS/MS
scan, they were allowed one repetition before a dynamic exclusion for a period
of 120s. External calibration of mass spectrometer was carried out using
reserpine and trypsinized bovine serum albumin (BSA) routinely.
Protein Identification and Relative Quantitation. The MS spectral data
obtained from 16 step gradient SCX fractions in each run were combined and
searched against non-redundant International Protein Index (IPI) database
(Rat v3.62, 39867 entries) from the European Bioinformatics Institute (EBI)
using ProteinpilotTM software 4.0.1 (Applied Biosystems). The following
parameters were selected: iTRAQ 4plex peptide labeled, trypsin digestion,
methyl methanethiosulfate (MMTS) alkylation cysteine residue, ID focus was
biological modifications, and FDR analysis.
Pro Group Algorithm calculates two types of scores for each protein: Unused
ProtScore and Total ProtScore. The Total ProtScore is based on all peptides
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pointing to a protein and is analogous to protein scores reported by other
protein identification softwares. The Unused ProtScore is a measurement of all
the peptides evidence for a protein that is not already explained by higher rank
proteins, and it is a true indicator of protein evidence, whereby Pro Group™
Algorithm distinguishes protein isoforms, protein subsets and suppresses false
positives. In the present study, a protein with “unused” confidence threshold
(ProtScore) >1.3% was reported and the corresponding False Discovery Rate
(FDR) was less than 1%.
Protein quantification was also performed by Proteinpilot software, which
calculated the relative abundance of iTRAQ labeled peptides and the
corresponding proteins by the intensity of the reporter ions (114 and 116). In
the present study, the threshold for protein differential expression was 3.0-fold.
Protein Identification and Relative Quantitation. The MS raw data were
analyzed essentially as described (1). In brief, ProteinPilot Software 4.0.1
(Applied Biosystems) was adopted to identify and quantify peptides and
proteins. The complete set of raw data files (*.wiff) were searched against the
non-redundant International Protein Index (IPI) database (Rat v3.62, 39867
entries) using the Paragon and ProGroup algorithms (Applied Biosystems). In
the present study, proteins with 95% or greater confidence as determined by
ProteinPilot™ Unused scores (≥1.3) were reported, and the corresponding
FDR was less than 1%. Based on individual peptide ratios, the relative
abundance of proteins was calculated. Details are provided in the
Supplemental Material.
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Bioinformatics Analysis of Differentially Expressed Proteins. The
bioinformatics analysis of the differentially expressed proteins was performed
with Ingenuity Pathways Analysis (IPA) software (version 6.3, Ingenuity
Systems, Redwood City, CA, http://www.ingenuity.com). The differentially
expressed proteins and the corresponding expression values were uploaded
into the software and assigned into different molecular and cellular functional
classes based upon the underlying biological evidence from the curated
Ingenuity Pathways Knowledge Base. Functional networks of these proteins
were algorithmically generated based on their connectivity and ranked
according to relevancy to the proteins in the input dataset. Canonical pathway
analysis identified the pathways, from the IPA library of canonical pathways,
which were most significant to the input dataset. The main biological functions
of the differentially expressed proteins were also collected from Uniprot protein
knowledge database (http://www.uniprot.org) and PubMed
(http://www.ncbi.nlm.nih.gov).
siRNA Transfection. siRNA transfections were performed according to the
manufacturer’s protocol (Sigma, Saint Louis, MO). Passage 2 HSCs cultured
in 24-well plates or 6-cm dishes were transfected at 50-70% confluence with
siRNAs targeting rat Bag2 and Bag3 by means of the siRNA transfection
reagent RNAiMAX (Invitrogen, Carlsbad, CA). NTC (Non-targeting control)
siRNA was transfected simultaneously as negative control. After 48 h
transfection, the efficiency of siRNA-mediated mRNA and protein degradation
was assessed by real-time quantitative polymerase chain reaction (qPCR) and
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Western blotting, respectively. qPCR analysis were performed with following
primers: bag2 sense, 5’ -AGGCAGATCAGCGATGGAGA- 3’, bag2 anti-sense,
5’ -AAGTGGCTCTTGGCATTCCC- 3’; bag3 sense, 5’
-CCGCACCTTTTCCATGCCTA- 3’, bag3 anti-sense, 5’
–ACTGAGATCGCTCAGGTCCG- 3’.
Cell Proliferation Assay. Forty-eight hours after siRNAs transfection, the cell
proliferation of HSCs was detected by incorporation of
5-ethynyl-2′-deoxyuridine (EdU) with the Click-iT EdU Cell Proliferation Assay
Kit (Invitrogen). Briefly, according to the kit's protocol, cells were incubated with
10 μM EdU for 16h before fixation, permeabilization, and EdU staining. EdU
was detected by Alexa Fluor 594 azide and nuclei were counterstained with
5 μg/ml Hoechst 33258 (Invitrogen). For each well, eight fields were randomly
counted at a magnification of 200. The results were expressed as the labeling
index according to the following formula: number of EdU-positive nuclei×100 /
number of total nuclei.
Cell Adhesion and Migration Assay. Untreated 96-well plates were coated
with 10 μg/ml collagen type I (Sigma) overnight at 37°C, then 1% BSA was
applied to block nonspecific adhesion. Normal HSCs, HSCs transfected with
siRNAs and NTC siRNA were seeded into triplicate wells of a 96-well plate
(5×104 cells/well) and allowed to attach for 30 min at 37°C. After careful wash
with pre-warmed PBS, cells were fixed with 2% formaldehyde and stained by
crystal violet (Sigma). To compare the number of attached cells, the stained
cells were lysed by 1% SDS and the OD value at 570 nm was recorded.
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Results were expressed as percentage change in the absorbance (OD 570 nm)
of adherent cells relative to NTC transfected cells.
The effects of siRNA transfecteion on HSCs migratory were measured by
using a modified Boyden chamber assay. Two days after transfection, 2 ×10 4
cells in serum-free DMEM were plated on the upper chamber of each
Transwell with 8μm pores (Costar, Corning Inc., NY). The low surface of
Transwell membranes were coated with collagen type I (Sigma), and the lower
chamber contained 800 μl completed medium. Transfected cells were
incubated for 16h at 37°C in 5% CO2. Non-migrating cells were removed from
the upper surface of the membrane with cotton swabs. Membranes were
stained with crystal violet and mounted onto glass slides, and migration was
quantified by counting cells in eight fields.
Supplemental Figure Legends
Supplemental Fig. 1. Identification of primary rat HSCs: Bunches of
cytoplasmic lipid droplets in D2 HSCs, (A) Hoffman Phase, (B) bright field, (C)
autofluorescence of cytoplasmic vitamin A lipid droplets, UV, (D)
autofluorescence of cytoplasmic vitamin A lipid droplets, FITC, (E) Sudan III
staining. Depletion of cytoplasmic lipid droplets in D10 HSCs, (F) Sudan III
staining (original magnification 200 ×). D2 and D10 HSCs represent day2
quiescent and day10 activated HSCs. Each image is a representative of three
independent experiments.
Supplemental Fig. 2 Culture activation of primary rat hepatic stellate cells
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(HSCs). Primary rat HSCs continuously expressed desmin, (A)
Immunofluorescent staining of desmin in D10 HSCs (FITC, green) (original
magnification 400 ×). Activated HSCs expressed α-smooth muscle actin
(α-SMA) and synthesized more collagen type I (COL I). (B) Immunofluorescent
staining of α-SMA in D10 HSCs (Texas red) (original magnification 400 ×). (C)
Immunofluorescent staining of COL I in D10 HSCs (Texas red) (original
magnification 400 ×). (D) The expression of α-SMA and COL I was determined
by Western blotting analyses using whole cell lysates, β-actin was served as a
loading control. D2 and D10 HSCs represent day2 quiescent and day10
activated HSCs. Each image is a representative of three independent
experiments.
Supplemental Fig. 3. Sirius red staining of liver sections. Liver cirrhosis was
induced by chronic intraperitoneal CCl4 administration (0.2 ml CCl4/ml of olive
oil, 412 mg CCl4/kg body weight, twice a week). Rats were sacrificed at day 56.
The formalin/paraffin sections of liver tissue were stained with Sirius red. The
collagen deposition was stained in red. (A) Normal control. (B) Experimental
model of rat liver cirrhosis. Each image is a representative of six animals in
each group (original magnification 100 ×).
Reference
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proteomics analysis of chondrogenic differentiation of C3H10T1/2
mesenchymal stem cells by iTRAQ labeling coupled with on-line
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two-dimensional LC/MS/MS. Mol Cell Proteomics 2010 Mar;9(3):550-564.
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