1
Supplementary Material and Methods
WST-1 cytotoxicity assay
HepG2 and BWTG3 cells were seeded in 96-well plates at 104 cells/well in
Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum and
incubated the next day with the indicated products (see cell culture) or 1% Triton X100. Cell viability was determined using the water-soluble tetrazolium salt (WST-1)
assay (Roche Diagnostics, Mannheim, Germany). The reagent was added, and the
cells were incubated for 4 h at 37°C. The absorbance of the bioreduced WST-1
(formazan) was measured at 450 nm against a background control at 655 nm
(Multiskan Ascent, Leuven, Belgium). Experiments were performed in sextuplicate.
This assay were applied to determine the optimal noncytotoxic concentration of
tunicamycin.
Caspase-3 activity assay
The enzymatic activation of effector caspase-3, a hallmark of apoptosis, was
evaluated in HCC cells following different treatments using the Caspase-3-Glo assay
(Promega) following the manufacturer’s instructions. Experiments were performed in
sextuplicate.
Proliferation assay
Proliferative activity was assessed by 5-bromo-2 ′ deoxyuridine (BrdU) labeling
(Roche, Mannheim, Germany) following the manufacturer's instructions. The
absorbance obtained in the ELISA was measured at 450 nm against a background
control at 690 nm (Multiskan Ascent, Leuven, Belgium). Experiments were performed
in sextuplicate.
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Total RNA extraction
Total RNA was extracted from all samples using the RNeasy Mini Kit (Qiagen,
Westburg BV, The Netherlands) with on-column DNAse treatment (Qiagen). Needle
homogenization was performed. The purity and quantity of total RNA was assessed
using spectrophotometry (Nanodrop; Thermo Scientific, Wilmington, USA). The ratio
of absorption at 260 and 280 nm was used to define RNA purity; samples with a
260:280 ratio between 1.8 and 2.0 were accepted.
Quantitative real-time PCR
One microgram of total RNA was converted to single strand cDNA by reverse
transcription (iScript, BioRad, California, USA) with oligo (dT) and random priming.
The cDNA was diluted 1/10 and used for real-time quantification using SYBR Green
(Sensimix, Bioline Reagents Ltd, London, UK) and 250 mM of each primer. A twostep program was run on a LightCyclerR 480 (Roche). Cycling conditions were 95°C
for 10 minutes and 45 cycles of 95°C for 10 seconds followed by 60°C for 1 minute.
Melting curve analysis confirmed primer specificities. All reactions were performed in
duplicate. Relative fold change was calculated using the ΔΔCT method. First, the
normalization of the threshold cycle (CT) values of the target gene was performed
with the CT of GAPDH in the same samples (ΔCT=CT target – CT GAPDH). The
expression was normalized again with the control (ΔΔCT=ΔCT – ΔCT control), and
the fold change was calculated (2-ΔΔCT). The PCR-efficiency of each primer pair was
calculated using a standard curve of reference cDNA. Amplification efficiency was
determined using the formula 10-1/slope. The primer set sequences are listed in Table
S1.
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Western blotting
Total protein extract was obtained by dissolving cells in RIPA buffer (1x PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS and 1× complete protease inhibitors
(Roche Diagnostics)). The total protein yield was determined using Bradford reagent
(Biorad). Approximately 25-50 µg of protein was separated by SDS-PAGE. The
proteins were transferred to a PVDF membrane (Millipore), which was subsequently
blocked and incubated with specific antibodies (Table S2) in 5% non-fat milk followed
by horseradish peroxidase-conjugated secondary antibodies. UPR-related antibodies
were validated using tunicamycin. To assess protein synthesis rate, HepG2 cells
were incubated with 1 µM puromycin (Sigma) for 30 minutes. The phosphorylation of
Ire1 was monitored by Phos-tag SDS gels following the manufacturer's instructions
(NARD Institute). Samples were separated by SDS-PAGE containing 50 µM Phostag (NARD Institute) and 50 µM MnCl. Gels were soaked in 1 mM EDTA for 10 min
before being transferred onto PVDF membranes. ECL detection reagent (Amersham
Life Science, New Jersey, USA) was used to visualize the specific proteins.
Quantification was performed using the NIH ImageJ software where band densities
were calculated and subtracted from the background.
Immunohistochemistry
Immunohistochemistry was performed on 5 μm sections of FFPE tissue by heatinduced epitope retrieval in 10 mM sodium citrate (pH=6) before blocking with 5%
BSA-PBS. Samples were incubated overnight at 4°C with antibodies for Chop (1:600
dilution), phospho-eIF2α (1:100 dilution) and Grp78 (1:400 dilution; Table S2).
EnVisionTM+ System-HRP (DAB) was applied as secondary antibody (Dako,
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Golstrup, Denmark), and sections were counterstained with hematoxylin. Staining
was semi-quantitatively measured by 2 independent observers (YV and DL) using
Olympus CellD software.
TUNEL immunofluorescence
Embedded liver sections were deparaffinized, rehydrated through graded alcohol,
and permeabilized with 0.1 % TritonX-100 at room temperature (8 min incubation).
Slides were rinsed twice in a phosphate-buffered saline (PBS). Apoptosis was
detected through in
situ terminal deoxynucleotidyl transferase (TdT)-mediated
deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL), using the In Situ Cell
Death Detection Kit (Roche, Vilvoorde, Belgium). Slides were incubated with the
TUNEL reaction mixture containing TdT and fluorescein-dUTP for 1 hour at 37°C;
thereafter, they were rinsed three times in PBS. The sections were mounted with an
antifade solution containing 4’,6-diamidino-2-phenylindole (DAPI) (Vectashield, Lab
Consult) for nuclear staining. Images were acquired on a Nikon TE300 inverted
epifluorescence microscope with a x20 objective and equipped with a Nikon DS-Ri1
cooled color CCD camera (Nikon Belux, Brussel, Belgium). For quantification of
apoptotic cells, five areas from each slide were examined and five slides per liver
were analyzed. Cells containing green fluorescence and either nuclear condensation
or chromatin fragmentation (without nuclear morphological changes) were identified
as apoptotic cells. Results were expressed as TUNEL-positive index (number of
TUNEL-positive cells per number of total cells quantified from DAPI-positive counts).
Transmission electron microscopy
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Murine liver fragments treated with saline for 30 weeks or with tunicamycin for 72 h or
liver tumors isolated from mice treated with DEN for 30 weeks were immersed in a
fixative solution of 2.5% glutaraldehyde and 4% formaldehyde in 0.1 M sodium
cacodylate buffer, placed in a vacuum oven for 30 min and left rotating for 3 h at
room temperature. This solution was later replaced with fresh fixative, and the
samples were left rotating overnight at 4°C. After washing, the samples were post
fixed in 1% OsO4 with K3Fe(CN)6 in 0.1 M sodium cacodylate buffer, pH 7.2. The
samples were dehydrated through a graded ethanol series, including a bulk staining
with 2% uranyl acetate at the 50% ethanol step followed by embedding in Spurr’s
resin. To select the area of interest on the block and to have an overview of the
phenotype, semi-thin sections were first cut at 0.5 mm and stained with toluidin blue.
Ultrathin sections of a gold interference color were cut using an ultra-microtome
(Leica EM UC6), followed by a post-staining with uranyl acetate and lead citrate in a
Leica EM AC20 and collected on Formvar-coated copper slot grids. The sections
were viewed with a transmission electron microscope (JEM 1010; JEOL, Tokyo,
Japan).
Choline positron emission tomography
In vivo tests were performed using positron emission tomography (PET). PET-CT
acquisitions were performed using a triple-modality Triumph II micro-PET/SPECT/CT
scanner (Gamma Medica-Ideas). This state-of-the-art scanner consists of a microPET module (LabPET8) with 2x2x10-mm LYSO/LGSO scintillators in an 8-pixel,
quad-APD detector module arrangement. This system can deliver a 1.4-mm spatial
resolution in rodents at a sensitivity of 4%, thereby covering a field-of-view of 10 cm
transaxially by 8 cm axially. The micro-CT portion consists of a high-resolution micro-
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CT tube with a focal spot size variable between 20 and 129 μm combined with a flatpanel CsI detector.
Animals (3 groups with n=3) were injected in the tail vein with 516 ± 25 µCi of
[18F]-fluoromethylcholine ([18F]FMCH) (Laboratory of Radiopharmacy, Ghent
University, Belgium) immediately prior to micro-PET scanning at the beginning of a
30-minute dynamic acquisition. For anatomical localization, a micro-CT scan was
sequentially acquired using 256 projections over 360 degrees at 75 kVp/240 μA and
1.3x magnification with a focal spot size of 50 μm. The resulting PET data were
reconstructed
using
30
iterations
of
the
Maximum-Likelihood
Expectation-
Maximization algorithm in a 160 x 160 x 63 matrix with a 0.5 x 0.5 x 1.175-mm voxel
size. No additional spatial filtering was applied. The acquired CT projection images
were reconstructed using a filtered back-projection algorithm. All images were fused
and analyzed using AMIDE software.
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Supplementary Tables
Table S1. Primers used for the qRT-PCR experiments. The PCR-efficiency of each primer pair was calculated using a
standard curve of reference cDNA. Amplification efficiency R² was determined using the formula 10^(-1/slope).
Gene
symbol
Reference
sequence
Species
Forward primer
Reverse primer
Efficiency
R2
Gapdh
NM_008084.2
Mus
musculus
GCCGGCTCAGTGAGACAAG
TGGCACCTTCAGCAACAATG
95.1
0.99
Atf4
NM_009716.2
Mus
musculus
GTTGAGCAGGAACGCAGTCT
T
GGCAGAAGAGCACTGATCG
TA
96
0.98
Chop
NM_007837.3
Mus
musculus
AGCGCAACATGACAGTGAAG
GTGTAATTCCAGGGGGAGG
T
101
0.99
Xbp1u
NM_013842.2
Mus
musculus
TCTCAAGCCGCCCCTCCGTT
GTGGCTGGCGTGCAAGGGA
T
107
0.97
Xbp1s
NM_013842.2
Mus
musculus
TCTCAAGCCGCCCCTCCGTT
CGGGGTTGCTGGTGTGCCA
T
97.6
0.98
Pdia4
NM_009787.2
Mus
musculus
ACGAGACCCCGGCGTTCGGA
TGGCACTTTGAGGAGGTGA
GCC
90.6
0.99
Grp78
NM_00116343
4.1
Mus
musculus
TGCCGAGCTAAATTACACATT
G
CCTTGTGGAGGGATGTACA
GA
107
0.99
Grp94
NM_011631.1
Mus
musculus
GAGGCGGCTCCTGAGACCG
AA
GGACCCTCATGGTGCGTGG
C
101.2
0.99
P58IPK
NM_008929.3
Mus
musculus
GCTGAGTGTGGAGTAAATGC
G
CGGCTGCGAGTAATTTCTTC
C
103
0.99
Gadd34
NM_008654.2
Mus
musculus
ACATGCGATATCCCGCGCGA
C
CGATCGTGGGTCCGGACTG
C
96.4
0.99
Gpx3
NM_008161.3
Mus
musculus
CCTTTTAAGCAGTATGCAGG
CA
CAAGCCAAATGGCCCAAGTT
98.5
0.99
Gclc
NM_010295.2
Mus
musculus
GGGAAGAGACCCAGCGCCA
C
GCACGTCCTTGTGCCGGTC
C
96.2
0.99
Ero1L
NM_015774.3
Mus
musculus
GGGGCCAGACGCTTGGAGG
A
CTCGCCCAGAAGCCAAAGG
C
97.1
0.99
Edem1
NM_138677.2
Mus
musculus
CGCGGAGACCCTTCCAATC
CCAATGTATCCAAGGCATCA
ACC
109
0.99
Canx
NM_00111049
9.1
Mus
musculus
CAACAGGGGAGGTTTATTTT
GCT
TCCCACTTTCCATCATATTTG
GC
101
0.99
Herpud1
NM_022331.1
Mus
musculus
ACGCCAAGTGTCGTTGTGTG
GTC
GCTCGACTGCGCTCAGGGA
TG
92.8
0.99
Erdj4
NM_013760.4
Mus
musculus
CGCCCTGTGGCCCTGACTTG
AGCTTTCAGGGGCAAACAG
CCA
98.1
0.98
GAPDH
NM_00125679
9.1
Homo
sapiens
TGCACCACCAACTGCTTA GC
GGCATGGACTGTGGT
CATGAG
91
0.99
ATF4
NM_001675.2
Homo
sapiens
GACCACGTTGGATGACACTT
G
GGGAAGAGGTTGTAAGAAG
GTG
97
0.99
CHOP
NM_00119505
3.1
Homo
sapiens
AAGGCACTGAGCGTATCATG
T
TGAAGATACACTTCCTTCTT
GAACA
105
0.99
NM_00107953
Homo
AGACAGCGCTTGGGGATGGA
CCTGCTGCAGAGGTGCACG
115
0.99
XBP1u
8
9.1
sapiens
T
TAG
XBP1s
NM_00107953
9.1
Homo
sapiens
AGACAGCGCTTGGGGATGGA
T
CCTGCACCTGCTGCGGACT
C
110
0.99
PDIA4
NM_004911.4
Homo
sapiens
TCCCATTCCTGTTGCCAAGAT GCCCTCGTAGTCTACAGCCT
99
0.99
GRP78
NM_005347.4
Homo
sapiens
GGGAACGTCTGATTGGCGAT
CGTCAAAGACCGTGTTCTCG
106
0.99
P58IPK
NM_006260.4
Homo
sapiens
TTTGCGTTCACAAGCACTTAA
C
GTTCTGCATCCCAAACACAA
AC
94
0.97
GADD34
NM_014330.3
Homo
sapiens
TCCTCTGGCAATCCCCCATA
GGAACTGCTGGTTTTCAGCC
109
0.99
ERO1L
NM_014584.1
Homo
sapiens
GCCAGGTTAGTGGTTACTTG
G
GGCCTCTTCAGGTTTACCTT
GT
108
0.99
ERDJ4
NM_012328.2
Homo
sapiens
GGTGTGCCAAAATCGGCATC
GCACTGTGTCCAAGTGTATC
ATA
100
0.98
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Table S2. Characteristics of the antibodies used in the study. The specificity, isotype, clone number, and catalog number of the
antibodies are indicated if provided.
Antigen
Antibody isotype, clone
Company
Cat no.
ATF4
Rabbit polyclonal IgG
Santa Cruz
sc-200
eIF2α
Rabbit polyclonal IgG
Cell Signaling
9721
Phospho-eIF2α
Rabbit monoclonal IgG, 119A11
Cell Signaling
3597
Phospho-PERK
Rabbit polyclonal IgG
Santa Cruz
sc-32577
CHOP
Mouse monoclonal IgG2a, L63F7
Cell Signaling
2895
GADD34
Rabbit polyclonal IgG, H193
Santa Cruz
sc-8327
IRE1
Rabbit polyclonal IgG, 14C10
Cell Signaling
3294
PDIA4
Rabbit polyclonal IgG
Cell Signaling
2798
GRP78
Rabbit monoclonal IgG, C50B12
Cell Signaling
3177
CD105
Goat polyclonal IgG
R&D systems
af1320
B-tubulin
Rabbit polyclonal IgG
Abcam
ab6046
Actin
Rabbit polyclonal IgG
Abcam
ab5694-100
GAPDH
Rabbit polyclonal IgG
Abcam
ab9485
Puromycin
Mouse monoclonal IgG1
KeraFAST
3RH11
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Supplementary Figure Legends
Fig. S1 Confirmation of the mouse model (A) Number of macroscopic tumors. (B)
Mean tumor burden. (C) Fibrosis as shown by mean Metavir-score. Data are
presented as the mean±SD of n=12. *p<0.05, **p<0.01, ***p<0.001. (D)
Representative images of a 25 week saline- or DEN-treated liver. Arrows indicate
tumors.
Fig. S2 Temporal dynamics of Nrf2-mediated genes (A) Real-time PCR analysis
of Gpx3 and (B) Gclc. Data are presented as the mean±SD of n=12. *p<0.05,
**p<0.01.
Fig. S3 Effect of UPR modulation on cell viability, proliferation and protein
synthesis in HCC cells (A) Protein synthesis rate as assessed by puromycin
incorporation in HepG2 cells. (B) Proliferation rate was assessed by measurement of
BrdU incorporation in HepG2 cells. (C) Cell viability of BWTG3 and Hepa1-6 cells, as
assessed by a WST-1 assay. For cytotoxic control, 1% Triton X-100 was applied. (D)
Effect of a PERK inhibitor on cell viability of HepG2 cells under ER stress in
combination with the indicated compounds for 48 hours. Experiments were repeated
twice with similar results. Data are presented as the mean±SD. *p<0.05, **p<0.01,
***p<0.001.
Fig. S4 The PERK inhibitor increased the hepatic apoptosis rate (A)
Representative TUNEL immunofluorescence and (B) quantification of the TUNELpositive index. *p< 0.05, **p<0.01.