1
2
3
4
Supplemental Materials
5
Construction of plasmids and preparation of adenoviral vectors
6
Supporting Materials and Methods
Rat cDNA encoding the Prdx5 gene was cloned across the BamH I–Sal I sites of
7
plasmid PGEX4T-3 and then into the pAdTrack- cytomegalovirus (CMV) shuttle
8
vector via KpnI–XbalI sites. The pAdTrack-CMV vector encodes enhanced green
9
fluorescent protein (EGFP) under the control of a separate CMV promoter to act as a
10
marker for transfection. The plasmid was linearized by restriction digestion with PmeI
11
and cotransformed into Escherichia coli BJ5183 cells with pAdEasy-1. Recombinants
12
were selected for kanamycin resistance and confirmed by restriction analysis. The
13
linearized recombinant plasmid was transfected into the adenovirus packaging cell
14
line HEK293 using lipofectamine in T-25 flasks according to manufacturer’s
15
instructions. Transfection and viral productions were monitored by green fluorescent
16
protein (GFP) expression. For viral purification, cells were harvested and subjected to
17
four freeze–thaw cycles in dry ice/methanol and centrifuged to obtain a crude viral
18
stock. The crude viral stock was further amplified and purified by CsCl banding. The
19
concentrations of Ad-Prdx5 and Ad-EGFP were determined by plaque-forming assay,
20
and expressed as plaque-forming units (pfu). Both vectors were diluted to 3×109
21
pfu/ml with 1 mL saline for intravenous injection into donor rats.
22
Experimental animals
23
Syngenic male Lewis rats (age, 8–12 weeks; body weight 250–340 g; Vital River
24
Experimental Animal, Beijing, China) were used in all experiments to exclude any
1
effects of immunologic interference. Rats were kept in the animal facilities at Nanjing
2
Medical University (Nanjing, China) and experiments were conducted in accordance
3
with the guidelines approved by the China Association of Laboratory Animal Care.
4
Experimental design and surgical procedure
5
The weights of the median liver lobes, comprising approximately 32.3% of the
6
weight of the entire liver of recipients (range, 28.3–36.1%), were selected as the grafts.
7
Small-for-size liver transplantation (SFSLT) was carried out with revascularization
8
but without reconstruction of the hepatic artery as described by Kamada (17) but with
9
slight modifications. The latter Prdx5 overexpression experiments were conducted in
10
three groups of rats: (i) Ad-Prdx5, (ii) Ad-EGFP control, and (iii) saline control.
11
Ad-Prdx5, Ad-EGFP or 1 mL 0.9% saline was injected via the penile vein into
12
prospective donor animals. Donor livers were harvested four days later.
13
Survival study
14
Fifteen rats both in the saline/Ad-EGFP control and Ad-Prdx5 treatment groups
15
with SFSLT were used for the survival study. Rats that lived for >7 days after
16
transplantation were termed “survivors”. The log-rank test was used to test the
17
equality of the three survival rates. A post-hoc multiple comparison of survival rates
18
between the three groups was conducted with a log-rank test, followed by a
19
Bonferroni correction. Statistical analyses were conducted using 13.0 SPSS computer
20
software (SPSS Incorporated, Chicago, IL, USA) and statistical significance inferred
21
at P<0.05.
22
Collection of specimens of liver tissue
1
Rats were killed for liver grafts 2, 6, 24, or 48 h after blood reperfusion (n=3 for
2
each group at each time point). Three liver tissues after ischemia for 1 h were
3
collected. Three liver tissues from sham operated rats were collected as control. Blood
4
samples collected from recipients after reperfusion were immediately centrifuged to
5
obtain serum (n=3 for each group at each time point). Serum samples were stored at
6
–80°C for subsequent analysis. Liver tissues were excised, weighed and processed for
7
further proteomic analysis. The remaining tissue was fixed and processed for
8
histology and immunohistochemical analysis.
9
Biochemical examination
10
Serum aspartate aminotransferase (AST) (n=3 for each group at each time point)
11
and alanine aminotransferase (ALT) (n=3 for each group at each time point) were
12
measured using a standard automatic Analyzer (Hitachi 7600-10; Hitachi
13
High-Technologies Corporation, Tokyo, Japan).
14
Protein extraction
15
Livers obtained from the rats at the above-mentioned six time points (three
16
independent rat groups for each time point) were homogenized in lysis buffer (7
17
M urea, 2 M thiourea, 4% [w/v] CHAPS, 2% [w/v] dithiothreitol DTT, and 2%
18
[v/v] IPG buffer [pH 3–10]) in the presence of 1% (v/w) protease inhibitor
19
cocktail (Pierce Biotechnology, Rockford, IL, USA). The mixture was placed on a
20
shaker at 4°C for 1 h, and insoluble matter subsequently removed by
21
centrifugation at 40 000 × g and 4°C for 1 h. The protein concentration in each
22
sample was determined by the Bradford method using bovine serum albumin
1
(BSA) as the standard.
2
Two-dimensional electrophoresis (2DE)
3
IPG strips (length, 24 cm; pH, 3–10 NL; GE Healthcare, San Francisco, CA,
4
USA) loaded with 120 µg of proteins extracted from the rat livers (three rats for
5
each time point) were rehydrated. After isoelectric focusing, the IPG strips were
6
equilibrated, run in an Ettan DALT twelve electrophoresis system (GE Healthcare)
7
and visualized by silver staining as described
8
generated from three independently run gels at each time-point (18 gels in total).
9
Statistical Analysis
(18)
. In this experiment, data were
10
The stained gels were scanned. The ImageMasterTM 2-D Platinum Software
11
(Version 5.0, Amersham Bioscience, Swiss Institute of Bioinformatics, Geneva,
12
Switzerland) was used for spot detection, quantification, and comparative
13
analyses as described
14
containing the independent protein groups and one containing a mixture of
15
these for each of the six time points. Thus, proteins from each time point were
16
repeated four times. The expression level was determined by the relative
17
volume of each spot in the gel and was expressed as:
(18)
. In this experiment, 18 gels were analyzed: three
18
19
% Volume = [spot volume/Σ volumes of all spots resolved in the gel]
20
21
The values obtained for 18 independent experiments were pooled for calculating
22
means and standard deviation. Protein spots differentially changed across time
1
points were determined if P<0.05 (one-way ANOVA).
2
Protein identification by matrix-assisted laser desorption/ionization-time of
3
flight (MALDI-TOF/TOF) mass spectrometry
4
Differential protein spots were excised, dehydrated in acetonitrile, and dried at
5
room temperature. Proteins were reduced using 10 mM DTT/25 mM NH4HCO3 at
6
56°C for 1 h and subsequently alkylated in situ with 55 mM iodoacetamide/25 mM
7
NH4HCO3 in the dark at room temperature for 45 min. Gel fragments were
8
thoroughly washed with 25 mM NH4HCO3, 50% acetonitrile, and 100% acetonitrile
9
and dried in a SpeedVac. Dried gel fragments were re-swollen with 2–3 μL trypsin
10
(Promoga, Madison, WI, USA) solution (10 ng/μL in 25 mM NH4HCO3) at 4°C for
11
30 min. Excess liquid was discarded and the gel plugs incubated at 37°C for 12 h.
12
Trifluoroacetic acid (TFA) at a final concentration of 0.1% was added to arrest the
13
digestive reaction.
14
The digests were immediately spotted onto 600-μm AnchorChips (Bruker
15
Daltonics, Bremen, Germany). Spotting was achieved by pipetting 1 μL of the analyte
16
onto the MALDI target plate in duplicate and subsequently adding 0.05 μL of 2
17
mg/mL α-HCCA in 0.1% TFA/33% acetonitrile containing 2 mM (NH4)3PO4. Bruker
18
peptide calibration mixture (Bruker Daltonics) was also spotted for external
19
calibration. All samples were air-dried at room temperature and 0.1% TFA used for
20
on-target washing. All samples were analyzed on a time-of-flight Ultraflex II mass
21
spectrometer (Bruker Daltonics) in positive-ion reflectron mode.
22
Each acquired mass spectrum (m/z range, 700–4000; resolution, 15000–20000)
23
was processed using the software packages FlexAnalysis v2.4 and Biotools 3.0
24
(Bruker Daltonics) with the following specifications: peak detection algorithm, Sort
25
Neaten Assign and Place (SNAP); S/N threshold, 3; and quality factor threshold, 50.
1
Trypsic autodigestion ion picks (842.51, 1045.56, 2211.10, and 2225.12 Da) were
2
used as internal standards for validating the external calibration procedure. Matrix
3
and/or autoproteolytic trypsin fragments were removed. The masses of the peptides
4
obtained were cross-referenced with the IPI rat database (41,251 sequences,
5
21,545,744 residues) by using Mascot (v2.1.03) in automated mode with the
6
following search parameters: a significant protein score at p<0.05; minimum mass
7
accuracy, 100 ppm; enzyme, trypsin; missed cleavage sites allowed, 1; cysteine
8
carbamidomethylation;
9
similarity between PI and relative molecular mass specified; and a minimum
10
acrylamide-modified
cysteine;
methionine
oxidation;
sequence coverage of 15%.
11
Protein identification was confirmed using the sequence information obtained
12
from MS/MS. Each acquired MS/MS spectrum was also processed using the software
13
packages FlexAnalysis v2.4 and Biotools 3.0 (Bruker Daltonics) by the SNAP
14
method at a signal-to-noise ratio threshold of 3.0. The MS/MS spectra were
15
cross-referenced with the IPI rat database using Mascot (v2.4) in automated mode
16
with the following search parameters: 100 ppm for the precursor ion and 0.3 Da for
17
the fragment ions. Cleavage specificity and covalent modifications were considered
18
as described above, and the score obtained was higher than the minimal significant
19
(P<0.05) individual ion score. All significant MS/MS identifications carried out using
20
Mascot were manually verified for spectral quality and matching the y and b ion
21
series. In the case of multiple entries corresponding to slightly different sequences,
22
only the database entry exhibiting the highest number of matching peptides was
23
included. Identified proteins are listed in Supplemental Table 1.
24
Cluster analysis
25
Identified 314 proteins were subjected to clustering analysis. For each
1
identified protein spot, mean abundance values from the three repeats were
2
calculated and normalized. The normalized abundance values were then loaded
3
into Cluster 3.0 software
4
algorithm with similarity metric of Euclidian distance. Different numbers of
5
clusters were tried, and the one with a minimal number of clusters and also
6
giving sufficient separation of expression patterns across time was finally
7
chosen. Clustering results were viewed using Tree-View software
8
Bioinformatics analysis using Pathway Studio software
(19)
, and the protein spots clustered using the k-Means
(20)
.
9
To further explore the significance of the proteins in each cluster, Pathway Studio
10
(v5.00) software (Ariadne Genomics Incorporated, MD, USA), a specialized graph
11
visualization engine, was used to determine the relevant molecular functions of
12
proteins exhibiting significantly differential expression during early period of rat SFS
13
liver transplantation. The gene list was imported into Pathway Studio to identify the
14
cellular processes influenced by these proteins. Each identified cellular process was
15
confirmed via the PubMed/Medline hyperlink embedded in each node.
16
Western blot analysis
17
Protein levels of vimentin, Grp78, Arhgdia, α-Snap, Prdx5, Sod1, Gapdh, PC,
18
β-actin, and α-tubulin in rat liver tissue at the six time points (sham group,
19
ischemia 1 h, reperfusion 2 h, 6 h, 24 h and 48 h post-transplantation) were
20
analyzed as described
21
Santa Cruz Technology, Incorporated, Santa Cruz, CA, USA), Arhgdia (SC-360,
22
diluted 1:1000; Santa Cruz Technology), α-tubulin (SC58666, diluted 1:500;
(18)
. Antibodies against PCB (SC-46228, diluted 1:500;
1
Santa Cruz Technology), vimentin (V5255, diluted 1:500; Sigma–Aldrich, St.
2
Louis, MO, USA), Gapdh (G9545, diluted 1:1000; Sigma–Aldrich), Sod1
3
(ab16831, diluted 1:500; Abcam, Cambridge, MA, USA), β-actin (ab6276, diluted
4
1:2000; Abcam), Grp78 (ab53068, diluted 1:1000; Abcam), α, β-Snap (ab50483,
5
diluted 1:1000; Abcam) and Prdx5 (ab16823, diluted 1:1000; Abcam) were used,
6
respectively. α-tubulin was used as the positive control.
7
Immunohistochemistry
Liver sections fixed in Bouin’s solution and embedded in paraffin were
8
9
immunostained as described
quenching
(21)
endogenous
. In brief, sections were incubated in 2% H 2 O 2
10
for
peroxidase
activity,
and
were
washed
in
11
phosphate-buffered saline (PBS). They were then blocked with a blocking serum
12
and incubated overnight at 4°C with primary antibodies against PCB (1:50),
13
Arhgdia (1:50), Prdx5 (1:100), vimentin (1:50), Grp78 (1:100) and α-Snap
14
(1:100). After washing thrice with PBS, sections were incubated with horseradish
15
peroxidase (HRP)-conjugated secondary antibodies. The immunoreactive sites
16
were visualized as brown staining with diaminobenzidine and mounted for
17
bright-field microscopy (Axioskop 2 plus, Zeiss, Oberkochen, Germany). Negative
18
controls were incubated with a solution devoid of primary antibody.
19
Apoptotic cells detected by fluorescent in-situ apoptosis
20
Frozen sections of liver grafts obtained at 24 h (n=3) and 48 h (n=3) were
21
examined for apoptotic cells using fluorescent terminal deoxynucleotidyl transferase
22
dUTP nick end labeling (TUNEL) assay according to the instructions of the
1
commercial kit (S7165, Chemicon International Incorporated, Temecula, CA, USA).
2
Ten random fields were counted for each TUNEL-stained tissue sample in a blinded
3
matter. Classic TUNEL positivity was characterized by focal nuclear staining. Three
4
different liver sections from different lobules were analyzed.
5
6
Supplemental Table 1
7
Identification of differentially expressed proteins using MALDI-TOF/TOF
8
9
Supplemental Figure 1
10
Six representative 2-DE maps of small-for-size liver grafts proteins at the 6 specific
11
time-points. Tissue proteins were extracted, separated by 2D-PAGE, and visualized by
12
silver-staining. PI and MW markers were labeled.
13
14
Supplemental Figure 2
15
(A) Representative western blot demonstrated a marked increase in Prdx5 protein
16
expression 6 h and 24 h after transplantation in the AdPrdx5 pretreatment group when
17
compared with controls. No significant differences in Prdx5 expression were observed
18
between the saline and AdEGFP control group. (B) Enhanced green fluorescence
19
protein (EGFP) expression was determined by fluorescent microscopy 6 h after
20
reperfusion. GFP-positive cells were detected in the AdPrdx5 treatment and AdEGFP
21
control groups; EGFP gene expression was not observed in the saline control group.