Supplementary Materials & Methods
Generation of Alb/AEG-1 mouse: To generate the Alb/AEG-1 transgene construct, the human
AEG-1 cDNA (containing a C-terminal HA-tag) was first excised from pcDNA3.1/AEG-1 with
BamHI and inserted into the EcoRI site in -globin exon 3 of the pBSpKCR3 vector. pBSpKCR3
contains part of exon 2, intron 2, and exon 3 of the rabbit -globin gene for efficient transgene
expression and has been described previously (1). A 2.3-kb NotI/BamHI fragment containing
the mouse albumin enhancer/promoter was then excised from pBS-KS(-)/Alb and inserted
between the SmaI and BamHI sites in -globin exon 2. pBS-KS(-)/Alb was kindly provided by
Dr. Snorri Thorgeirsson (NCI) and contains an upstream enhancer region (–10400 to –8500)
fused to the 335-bp core promoter (2). The sequences of both the AEG-1 cDNA and the
albumin enhancer/promoter in the Alb/AEG-1 transgene construct were verified by automated
DNA sequencing. Microinjection and manipulation procedures were performed according to
standard procedures in the Massey Cancer Center Transgenic/Knockout Mouse Core. These
studies were approved by the Institutional Animal Care and Use Committee at Virginia
Commonwealth University. To generate Alb/AEG-1 transgenic mice, a 5.3-kb XhoI fragment
was excised from the Alb/AEG-1 construct and microinjected at a concentration of 2 ng/l into
the male pronucleus of single-cell mouse embryos obtained from mating B6CBAF1/J (C57BL/6
× CBA) males and females. The injected embryos were then reimplanted into the oviducts of
pseudopregnant CD-1 female mice. The resulting offspring were screened for the presence of
the Alb/AEG-1 transgene by PCR analysis of genomic tail DNA using a human AEG-1 cDNA
primer: 5’-GAAGACCTTCCAGTGAATACCTC-3’ (sense) and a -globin exon 3 primer: 5’ATGTCCTTCCGAGTGAGAGACAC-3’ (anti-sense). Amplification of DNA from potential
founders carrying the Alb/AEG-1 transgene generated a 587-bp PCR product. The hepatocyte-
1
specific expression of AEG-1 was confirmed by Western blot in multiple tissues using anti-HA
and anti-AEG-1 antibodies.
Induction of chemical carcinogenesis: For induction of chemical carcinogenesis, a single i.p.
injection of 10 g/gm body weight of N-nitrosodiethylamine (DEN) was given at 14 days of age
to male WT and Alb/AEG-1 mice (3). The animals were sacrificed at 28 weeks of age and liver,
internal organs and blood were collected. Serum liver enzymes were analyzed in the Molecular
Diagnostic Laboratory, Department of Pathology, VCU using standard procedures.
Cell culture, viability and transfection assays: Primary mouse hepatocytes were isolated
from WT and Alb/AEG-1 mice in the Cell and Molecular Biology Core in VCU as described
previously (4) and were cultured in Williams E medium containing NaHCO3, L-glutamine, insulin
(1.5 M) and dexamethasone (0.1 M). Growth factors were not added when the cells were
cultured in basal media. For chemoresistance assay mouse hepatocytes were cultured in 100
nM 5-FU or 5 nM Doxorubicin. Primary human hepatocytes were obtained from the Liver Tissue
Cell Distribution System (LTCDS) (NIH contract #N01-DK-7-0004 / HHSN267200700004C) and
were cultured in Hepatocyte Culture Medium (HCM) containing the supplements (Lonza).
Human umbilical vein endothelial cells (HUVEC) were obtained from Lonza and were cultured
according to the provided protocol. Cell viability was determined by standard MTT assay as
described (5). Control siRNA and siRNA for mouse TFF3 and FXII were obtained from Santa
Cruz Biotechnology and were transfected into hepatocytes using Promofectin-siRNA
transfection reagent (PromoCell GmbH) according to the manufacturer’s instruction. The
siRNAs used are pools of three different siRNAs targeting each gene.
Generation of Lenti.AEG-1: For lentivirus vector production, 293T cells were co-transfected
with plasmids bearing the lentiviral genome (CMVR8.91), an envelope coding viral coat protein
(VSVG) and the transfer vector constructs bearing sequence encoding AEG-1 and IRES-GFP or
2
IRES-GFP only. Virions were harvested 40 hr post-transfection and concentrated by
ultracentrifugation. Titers were determined on 293T cells as transducing units (tu) using serial
dilutions of vector stocks with 8 g/ml polybrene (Sigma, St. Louis, MO) (6).
Polysome purification, total RNA extraction, real time PCR and microarray assay:
Purification of polysomal fractions from WT and Alb/AEG-1 hepatocytes was performed exactly
as described (7). Total RNA was extracted from each polysomal fraction as well as from WT
and Alb/AEG-1 livers using Qiagen miRNAeasy mini kit (Qiagen). Real time PCR was
performed using an ABI ViiA7 fast real time PCR system and Taqman gene expression assays
according to the manufacturer’s protocol (Applied Biosystems). The data were normalized by
GAPDH expression level for mRNAs and U6 snRNA expression level for miRNA. An Affymetrix
oligonucleotide microarray (GeneChip® Mouse Genome 430A 2.0 Array representing
approximately 14,000 well-characterized mouse genes) analysis was performed to compare
gene expression between DEN-treated WT and Alb/AEG-1 liver samples. The standard
Affymetrix® protocol was used for generation of fragmented labeled cRNA for hybridization.
Every chip was scanned at a high resolution, with pixelations ranging from 2.5 µm down to 0.51
µm, by the Affymetrix GeneChip® Scanner 3000 according to the GeneChip® Expression
Analysis Technical Manual procedures (Affymetrix, Santa Clara, CA). After scanning, the raw
intensities for every probe were stored in electronic files (in .DTT as well as in .DAT and .CEL
formats) by the GeneChip® Operating Software (GCOSv1.4). Several parameters were
monitored for hybridization quality control purposes, such as the average background (Bkg), the
scaling factor (SF), which was used to scale all probes sets to a target value (TGT) set at 100;
the noise (RawQ) values; the percentage of present called probe sets (%P) and the 3’/5’ ratios
of the signal intensity values for two housekeeping genes, GAPDH and -actin, as well as
exogenous spike controls such as, Bacillus subtilis genes that are absent in eukaryotic samples
3
(lys, phe, thr, and dap). Normalization, background subtraction and expression values for every
probe set was calculated using the method by Irizarry RA et al., which is an effective expression
summary motivated by a log scale linear additive model (8). This summary statistics is referred
to as the log scale robust multi-array analysis (RMA). To identify probe sets that were
significantly different among the different sample types analyzed in this study, we performed t/Ftests separately for each probe set using the normalized log2-intensities. To adjust for multiple
hypothesis testing, the resulting p-values was used to obtain the false discovery rates (FDR)
using the q-value method. Genes were considered to have a significantly different expression
levels based on a p-value of <0.05%. All analyses were performed on the R environment using
functions provided by the BioConductor packages. Unsupervised learning methods, specifically
hierarchical clustering using Pearson (centered) correlation, were performed using the BRBArrayTools v3.1.0 Excel add-in, which provided further graphical representation of classification
results.
Capillary-like tube formation assay: Tube formation by human umbilical vein endothelial cells
(HUVEC) was performed using Cultrex basement membrane extract (R&D Systems) as
described (9). HUVECs were seeded in 96-wells plates at 4 × 104 cells/well, treated with the
condition medium collected from WT and Alb/AEG-1 hepatocytes and incubated at 37°C for ~6
h. Images were captured using an inverted microscope (Nikon) and the degree of network
formation was quantified using an Image analyzer (National Institutes of Health Image).
Chicken chorioallantoic membrane (CAM) assay: Conditioned media from WT and Alb/AEG1 hepatocytes were implanted on the CAM surface of 9-day-old chick embryos according to
established protocols (9). One week after inoculation, the neovasculature was examined and
photographed. Angiogenesis was quantified by counting the blood vessel branch points under a
4
stereomicroscope. The angiogenic index was calculated by subtracting the number of branch
points from the branching in the control group.
Immunohistochemistry: Immunohistochemistry was performed using formalin-flixed paraffinembedded (FFPE) sections as described previously (5). The sections were blocked in PBST
using 10% normal goat serum for rabbit polyclonal antibody, 10% normal horse serum for
mouse monoclonal antibody and 10% normal rabbit serum for goat polyclonal antibody. The
primary antibodies were diluted in PBST containing 5% corresponding blocking serum. The
primary antibodies used were: AFP (Santa Cruz; rabbit polyclonal; 1:200); TSPAN8 (Abnova;
mouse monoclonal; 1:200); SCD2 (Santa Cruz, goat polyclonal; 1:50); TFF3 (Santa Cruz; goat
polyclonal; 1:200); LCN2 (Santa Cruz; rabbit polyclonal: 1:150); LPL (Santa Cruz; rabbit
polyclonal; 1:200); MEOX2 (Santa Cruz; mouse monoclonal; 1:150); CD31 (Dako; mouse
monoclonal; 1;100). Secondary antibodies were diluted in PBST containing corresponding 2.5%
blocking serum. The signals were developed by avidin-biotin-peroxidase complexes with a DAB
substrate solution (Vector laboratories). Images were analyzed using an Olympus microscope.
Immunofluorescence and Western blot analyses: Immunofluorescence and Western blot
analyses were performed as described (5). For immunofluorescence the primary antibodies
were -H2AX (Millipore; mouse monoclonal; 1:500) and AEG-1 (chicken polyclonal; 1:500) and
the secondary antibodies were Alexa488-conjugated anti-mouse IgG (Molecular Probes; 1:400)
and Alexa568-conjugated anti-chicken IgG (Molecular Probes; 1:400), respectively. The slides
were mounted in VectaShield fluorescence mounting medium containing 4,6 -diamidino-2phenylindole (Vector Laboratories). Images were analyzed using a Zeiss confocal laser
scanning microscope. For Western blot, the primary antibodies were AEG-1 (chicken polyclonal;
1:500), HA (Sigma; chicken polyclonal; 1:2000), ATM (Cell Signaling; rabbit polyclonal; 1:800);
phospho-ATM (Novus Biological; mouse monoclonal; 1:2000); ATR (Cell Signaling; rabbit
5
polyclonal; 1:800); phospho-ATR (Cell Signaling; rabbit polyclonal; 1:800); CHK1 (Santa Cruz;
mouse monoclonal; 1:200); phospho-CHK1 (Cell Signaling; rabbit polyclonal; 1:1000); CHK2
(Santa Cruz; mouse monoclonal; 1:200); phospho-CHK2 (Cell Signaling; rabbit polyclonal;
1:1000); p53 (Cell Signaling; mouse monoclonal; 1:1000); phospho-p53 (Cell Signaling; rabbit
polyclonal; 1:1000); p21 (Cell Signaling; rabbit polyclonal; 1:1000); p38 MAPK (Cell Signaling;
rabbit polyclonal; 1:1000); phospho-p38 MAPK (Cell Signaling; rabbit polyclonal; 1:1000); p16
(Cell Signaling; rabbit polyclonal; 1:1000); ERK (Cell Signaling; rabbit polyclonal; 1:2,000);
phospho-ERK (Cell Signaling; rabbit polyclonal; 1:2,000); AKT (Cell Signaling; rabbit polyclonal;
1:1000); phospho-AKT (Cell Signaling; rabbit polyclonal; 1:1000); phospho-EGFR-Y1068 (Cell
Signaling; rabbit polyclonal; 1:700); EGFR (Cell Signaling; rabbit polyclonal; 1:700); Fatty Acid
Synthase (Cell Signaling; rabbit polyclonal; 1:1000); Acetyl Co-A Carboxylase (Cell Signaling;
rabbit polyclonal; 1:1000); EF1 (Millipore; mouse monoclonal; 1: 1000).
Assay for Senescence Associated -galactosidase (SA--gal) activity: Cells were cultured
for six days and SA--gal-positive cells were detected using Senescence Detection Kit
(Biovision) according to the manufacturer’s protocol.
Measurement of reactive oxygen species (ROS): The day before experiment, 1X104
cells/well were plated in a 96-well plate & total ROS was detected using Total ROS/Superoxide
Detection Kit (Enzo Life Sciences) according to the manufacturer’s protocol using a
fluorescence plate reader with excitation at 490 nm and emission at 525 nm. The values were
normalized by background fluorescence and further by respective unstained values. Each
sample was measured using 6-10 replicates and the assay was performed in 4 different sets of
animals.
Mass spectrometric analysis of conditioned media: Sample preparation and digestion for
mass spectrometry: Conditioned media from WT and Alb/AEG-1 hepatocytes were collected
6
one day after isolation and concentrated using Amicon Ultra-15 Centrifugal Filter Units
(Millipore). Protein concentration was determined by standard Bradford assay. One hundred g
of protein from each sample were subjected to electrophoresis using a 4-12% NuPAGE gel
(Invitrogen). The gel was stained with SimplyBlue Safe Stain (Invitrogen), dissected into pieces
and was dehydrated with acetonitrile for 5-10 minutes. Samples were incubated for 30 minutes
at 37°C in 10 mM DTT. Iodoacetamide was added to a final concentration of 100 mM and
incubated for an additional 30 minutes at room temperature in the dark and dehydrated again in
acetonitrile. Samples were rehydrated in 100 mM ammonium biocarbonate for 10 minutes,
followed by dehydration in acetonitrile and rehydration in trypsin (1:50 enzyme to substrate
ratio) at room temperature for 10 min. Samples were incubated in 50 mM ammonium
biocarbonate overnight at 37°C. The reaction was then stopped with extraction buffer (50%
acetonitrile; 5% formic acid) and dried using a vacuum centrifugation and stored at -80 °C prior
to mass spectrometric analysis. Mass spectrometry: The peptides were resuspended in 10 μL
of 3% acetonitrile and 0.5% formic acid and vortexed before LC-MS/MS analysis (Agilent 1100,
(LTQ Orbitrap XL by Thermo Electron). A PicoFrit column (New Objective, Woburn, MA), with
an inner diameter of 75 μm packed with 12-14 cm of ReproSil-Pur C18 3 μm particles, was
directly interfaced to an LTQ Orbitrap XL mass spectrometer equipped with a custom nanoelectrospray ionization source. 4 μL of the peptide mixture was injected and separated by a 90
min gradient (~0.7%B/min.) of increasing acetonitrile from 5 - 60%. Two replicate MS analysis
was carried out per sample. MS analysis settings for protein identification were as follows. One
precursor MS scan at 60,000 resolution in profile mode was followed by data-dependent scans
of the top 8 most abundant ions at low-resolution in centroid mode. Dynamic exclusion was
enabled with a repeat count of 2, repeat duration of 20 seconds, exclusion duration of 30
seconds and an exclusion list size of 500. MS/MS spectra were collected with normalized
collision energy of 28 and an isolation width of 3 amu. Mass spectrometry data analysis: All
7
discovery data was processed using Agilent Spectrum Mill MS Proteomics Workbench (Agilent
Technologies, Palo Alto, CA) v4.0 beta. MS/MS spectra were searched against the Mouse
International Protein Index (IPI) database (version 3.48) with parent mass tolerance of 20 ppm,
fragment
mass
tolerance
0.7
Da,
a
maximum
of
two
missed
cleavages,
and
carbamidomethylation and oxidized methionines/pyroglutamic acid as fixed and variable
modifications, respectively. Database matches for individual spectra were auto validated
according to user-defined scoring thresholds for both peptides and proteins in a two-step
process. No manual inspection of spectra or validation was performed; however peptide false
discovery rate was minimized by using strict scoring criteria (see next paragraph). For protein
auto validation (step 1), auto validation criteria included a cumulative score of ≥ 25 based upon
individual scores of multiple peptides derived from a given protein. Peptide scores in protein
mode had to be ≥ 10 with a scored peak intensity (SPI) of ≥ 70% for peptides with a precursor
charge state of +2. Scored peak intensity refers to the percentage of the annotated MS/MS
spectrum that is explained by the database match. Peptides with precursor charges of +3 and
+4 had to meet scoring thresholds of ≥ 13 and 70% SPI. For peptide auto validation (step 2),
single peptides derived from a given protein had to meet scoring thresholds of ≥ 13 and ≥ 70%
SPI for all charge states. In both auto validation steps, the delta rank1 – rank2 threshold was >
2. In calculating scores at the protein level and reporting the identified proteins, redundancy is
addressed in the following manner: the protein score is the sum of the scores of distinct
peptides. A distinct peptide is the single highest scoring instance of a peptide detected through
an MS/MS spectrum. MS/MS spectra for a particular peptide may have been recorded multiple
times, (i.e. as different precursor charge states, isolated from adjacent reverse phase fractions,
oxidation of Met) but are still counted as a single distinct peptide. In Spectrum Mill, false
discovery rates (FDRs) are calculated at 3 different levels: spectrum, distinct peptide, and
distinct protein. Peptide FDRs are calculated in Spectrum Mill using essentially the same
8
pseudo-reversal strategy evaluated by Elias, et al. (10) and shown to perform the same as
concatenation. A false distinct protein ID occurs when all of the distinct peptides, which group
together to constitute a distinct protein, have a deltaForwardReverseScore ≤ 0. We adjusted
settings to provide peptide FDR of ≤1.5%. Spectrum Mill also carries out protein grouping using
the methods as described (11). Briefly, when a peptide sequence >8 residues long is contained
in multiple protein entries in the sequence database, the proteins are grouped together and the
highest scoring one and its accession number are reported. In some cases when the protein
sequences are grouped in this manner there are distinct peptides which uniquely represent a
lower scoring member of the group (isoforms and family members). Each of these instances
spawns a subgroup and multiple subgroups are reported and counted towards the total number
of proteins. Label-free, relative quantitation: The sum of the precursor-ion signal intensities of
all peptides derived from each protein was used as an approximation of that protein’s
expression level across the three sample categories. The peak area for the extracted ion
chromatogram of each precursor ion in the intervening high-resolution MS1 scans of the datadependent LC-MS/MS runs was calculated automatically by the Spectrum Mill software using
narrow windows around each individual member of the isotope cluster. Peak widths in both the
time and m/z domains were dynamically determined based on MS scan resolution, precursor
charge and m/z subject to quality metrics on the relative distribution of the peaks in the isotope
cluster vs. theoretical. Total intensity information for all proteins across the samples was
exported to Excel where ratios of the total intensities were calculated. Since an equivalent
amount of protein (2g) for a given sample was loaded onto column for LC-MS/MS analysis,
prior to ratio calculations all protein intensities were normalized by the total ion current (TIC) as
reported by Spectrum Mill.
Analysis of monoubiquitination of AEG-1: Hep-pc-4 (control HepG3 clone) and Hep-AEG-114 cells (HepG3 cells stably overexpressing AEG-1) were treated or not with proteasome
9
inhibitor MG-132 (5 M; Tocris Biosciences) for 12 hours. The lysates were subjected to
immunoprecipitation using anti-ubiquitin antibody ((Cell Signaling; mouse monoclonal; 1:200)
and immunoblotting using anti-AEG-1 antibody as described (12). Five per cent of total cell
lysate was used as input.
Statistical analysis: Data were represented as the mean  Standard Error of Mean (S.E.M)
and analyzed for statistical significance using one-way analysis of variance (ANOVA) followed
by Newman-Keuls test as a post hoc test. A P value of < 0.05 was considered as significant.
10
Supplementary legends
Table S1. Fold-changes in gene expression in Alb/AEG-1 liver when compared to that in WT
liver after DEN treatment. mRNAs that show at least two-fold change in Alb/AEG-1 hepatocytes
compared to WT hepatocytes are displayed.
Table S2. Fold-changes in protein expression in CM from Alb/AEG-1 hepatocytes when
compared to that from WT hepatocytes. Proteins that are represented by at least two unique
peptides and that show at least two-fold increase in Alb/AEG-1 hepatocytes compared to WT
hepatocytes are displayed. The components of the coagulation pathway are highlighted in red.
Fig. S1. Abnormalities in hepatic functions in DEN-treated Alb/AEG-1 mice. Serum levels of
aspartate aminotransferase (AST) (A), alanine aminotransferase (ALT) (B) and alkaline
phosphatase (Alk Phos) (C) in WT (n = 11) and Alb/AEG-1 (n = 17) mice were analyzed. The
data represent mean ± SEM. *: p<0.01.
Fig. S2. Alb/AEG-1 mice develop more aggressive tumors at 32 weeks when compared to WT
mice. Photomicrograph of livers of mice (WT: n = 7; Alb/AEG-1: n = 9) treated with DEN at 32
weeks of age. The nodules in Alb/AEG-1 mice are much larger than those of WT mice. The
asterisk indicates a markedly large nodule in Alb/AEG-1 mice.
Fig. S3. Supervised samples and genes cluster analysis of genes whose expression is altered
in DEN-treated Alb/AEG-1 livers when compared to DEN-treated WT livers. RNA was analyzed
using Affymetrix oligonucleotide microarrays. Two-dimensional hierarchical clustering of
samples and genes was performed using Pearson (centered) correlation and average linkage,
based on the 25 probe sets that were significantly different between DEN-treated WT and
Alb/AEG-1 livers. Each of the 25 rows in the heat map located beneath the dendrogram shows
the relative expression for that specific probe set in the separate 6 samples (columns). The
11
relative probe set expression levels are plotted according to the color scale at the bottom of the
heat map, where red and green areas correspond to probe sets overexpressed and
underexpressed, respectively, compared to the median intensity across the 6 samples.
Fig. S4. Gene expression changes in DEN-treated Alb/AEG-1 mice. A. Analysis of mRNA levels
of the indicated genes by real-time PCR using five animals/group. The most robust induction
was observed for -feto protein (Afp), a known marker for HCC (13), and for Tspan8,
tetraspanin 8, which mediates tumor invasion, angiogenesis and metastasis (14). Tspan8 has
also been identified as an AEG-1-induced gene in in vitro systems using human HCC cells and
it is overexpressed in human HCC indicating that it might play an important role in mediating
AEG-1-induced hepatocarcinogenesis (5, 15). Scd2, encoding stearoyl co-A desaturase, a
lipogenic enzyme necessary for synthesis of monounsaturated fatty acids; Lpl, encoding
lipoprotein lipase; and Apoa4 and Apoc2, encoding apolipoproteins A4 and C2, respectively,
might play significant role in conferring the steatotic phenotype observed in Alb/AEG-1 mice.
Lcn2, encoding lipocalin 2, promotes invasion and metastasis and is induced by AEG-1 (16).
Tff3, encoding trefoil factor 3, a pro-angiogenic factor promoting invasion and metastasis and
also overexpressed in HCC, is induced by AEG-1 (17, 18). In contrast, Meox2, a tumor
suppressor gene with potent anti-angiogenic properties, is inhibited by AEG-1 (19). The Y-axis
represents fold change in mRNA level when the expression level in WT mice was regarded as
1. The data represent mean ± SEM. *: p<0.01. B. Immunohistochemical analysis of the
indicated proteins in the sections of livers from WT and Alb/AEG-1 mice.
Fig. S5. AEG-1 is overexpressed in hepatocytes isolated from Alb/AEG-1 mice. AEG-1
expression was detected by Western blot analysis using hepatocytes isolated from WT and
Alb/AEG-1 mice with anti-AEG-1 and anti-HA antibodies. -tubulin was used as loading control.
12
Fig. S6. AEG-1 protects hepatocytes from senescence. A. Analysis of senescence-associated
markers in WT and Alb/AEG-1 hepatocytes. Cathepsin D and eEF1A1, two senescenceassociated markers (20), were significantly upregulated and downregulated, respectively, in WT
hepatocytes versus Alb/AEG-1 hepatocytes. B. Primary human hepatocytes were transduced
with a lentivirus expressing GFP (Lenti.GFP) or expressing AEG-1 (Lenti.AEG-1). At day 6,
Lenti.GFP-transduced primary human hepatocytes were vacuolated and >60% cells stained
positive for SA -gal while Lenti.AEG-1 transduced cells were viable and none of the cells were
SA -gal positive.
Fig. S7. TFF3 and FXII are efficiently knocked down by the corresponding siRNAs in Alb/AEG-1
hepatocytes. Alb/AEG-1 hepatocytes were either mock transfected or transfected with either
control siRNA (siCon) or with siRNA for TFF3 or FXII. TFF3 (A) and FXII (B) mRNA levels were
detected by real-time PCR and normalized by GAPDH mRNA level. The data represents mean
± SEM. *: p<0.05.
Fig. S8. AEG-1 does not change mature miR-181a level. miR-181a level was determined by
real-time PCR in WT and Alb/AEG-1 hepatocytes obtained from four independent pairs of
animals. The Y-axis represents fold change in miR-181a level when the expression level in WT
hepatocytes was regarded as 1.
Fig. S9. Overexpressed AEG-1 is monoubiquitinated. Hep-pc-4 (control HepG3 clone) and
Hep-AEG-1-14 cells (HepG3 cells stably overexpressing AEG-1) were treated or not with MG132. The lysates were subjected to immunoprecipitation using anti-ubiquitin antibody and
immunoblotting using anti-AEG-1 antibody. Five per cent of total cell lysate was used as input.
13
Fig. S10. Enzymes regulating fatty acid synthesis are overexpressed in Alb/AEG-1 mice.
Lysates from WT and Alb/AEG-1 livers were subjected to Western blot analysis for the indicated
proteins.
References:
1.
Howes KA, Ransom N, Papermaster DS, Lasudry JG, Albert DM, Windle JJ. Apoptosis
or retinoblastoma: alternative fates of photoreceptors expressing the HPV-16 E7 gene in the
presence or absence of p53. Genes Dev 1994;8:1300-1310.
2.
Ramirez MI, Karaoglu D, Haro D, Barillas C, Bashirzadeh R, Gil G. Cholesterol and bile
acids regulate cholesterol 7 alpha-hydroxylase expression at the transcriptional level in culture
and in transgenic mice. Mol Cell Biol 1994;14:2809-2821.
3.
Murakami H, Sanderson ND, Nagy P, Marino PA, Merlino G, Thorgeirsson SS.
Transgenic mouse model for synergistic effects of nuclear oncogenes and growth factors in
tumorigenesis: interaction of c-myc and transforming growth factor alpha in hepatic
oncogenesis. Cancer Res 1993;53:1719-1723.
4.
Bissell DM, Guzelian PS. Degradation of endogenous hepatic heme by pathways not
yielding carbon monoxide. Studies in normal rat liver and in primary hepatocyte culture. J Clin
Invest 1980;65:1135-1140.
5.
Yoo BK, Emdad L, Su ZZ, Villanueva A, Chiang DY, Mukhopadhyay ND, Mills AS, et al.
Astrocyte elevated gene-1 regulates hepatocellular carcinoma development and progression. J
Clin Invest 2009;119:465-477.
14
6.
Kock N, Kasmieh R, Weissleder R, Shah K. Tumor therapy mediated by lentiviral
expression of shBcl-2 and S-TRAIL. Neoplasia 2007;9:435-442.
7.
Yoo BK, Chen D, Su Z-Z, Gredler R, Yoo J, Shah K, Fisher PB, et al. Molecular
mechanism of chemoresistance by Astrocyte Elevated Gene-1 (AEG-1). Cancer Res
2010;70:3249-3258.
8.
Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of
Affymetrix GeneChip probe level data. Nucleic Acids Res 2003;31:e15.
9.
Chen D, Yoo BK, Santhekadur PK, Gredler R, Bhutia SK, Das SK, Fuller C, et al.
Insulin-like Growth Factor-Binding Protein-7 Functions as a Potential Tumor Suppressor in
Hepatocellular Carcinoma. Clin Cancer Res 2011;17:6693-6701.
10.
Elias JE, Gibbons FD, King OD, Roth FP, Gygi SP. Intensity-based protein identification
by machine learning from a library of tandem mass spectra. Nat Biotechnol 2004;22:214-219.
11.
Nesvizhskii AI, Aebersold R. Interpretation of shotgun proteomic data: the protein
inference problem. Mol Cell Proteomics 2005;4:1419-1440.
12.
Yoo BK, Santhekadur PK, Gredler R, Chen D, Emdad L, Bhutia SK, Pannell L, et al.
Increased RNA-induced silencing complex (RISC) activity contributes to hepatocelllular
carcinoma. Hepatology 2011;53:1538-1548.
13.
Donati M, Brancato G, Donati A. Clinical biomarkers in hepatocellular carcinoma (HCC).
Front Biosci (Schol Ed) 2010;2:571-577.
14.
Zoller M. Tetraspanins: push and pull in suppressing and promoting metastasis. Nat Rev
Cancer 2009;9:40-55.
15.
Kanetaka K, Sakamoto M, Yamamoto Y, Yamasaki S, Lanza F, Kanematsu T, Hirohashi
S. Overexpression of tetraspanin CO-029 in hepatocellular carcinoma. J Hepatol 2001;35:637642.
15
16.
Rodvold JJ, Mahadevan NR, Zanetti M. Lipocalin 2 in cancer: When good immunity goes
bad. Cancer Lett 2012;316:132-138.
17.
Okada H, Kimura MT, Tan D, Fujiwara K, Igarashi J, Makuuchi M, Hui AM, et al.
Frequent trefoil factor 3 (TFF3) overexpression and promoter hypomethylation in mouse and
human hepatocellular carcinomas. Int J Oncol 2005;26:369-377.
18.
Rodrigues S, Van Aken E, Van Bocxlaer S, Attoub S, Nguyen QD, Bruyneel E, Westley
BR, et al. Trefoil peptides as proangiogenic factors in vivo and in vitro: implication of
cyclooxygenase-2 and EGF receptor signaling. FASEB J 2003;17:7-16.
19.
Patel S, Leal AD, Gorski DH. The homeobox gene Gax inhibits angiogenesis through
inhibition of nuclear factor-kappaB-dependent endothelial cell gene expression. Cancer Res
2005;65:1414-1424.
20.
Byun HO, Han NK, Lee HJ, Kim KB, Ko YG, Yoon G, Lee YS, et al. Cathepsin D and
eukaryotic translation elongation factor 1 as promising markers of cellular senescence. Cancer
Res 2009;69:4638-4647.
16