1
Supporting Materials and Methods
2
Reagents
3
Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS)
4
were purchased from Gibco-BRL (Invitrogen). Antibodies against CypB and HIF-1α were
5
purchased from Abcam and BD Biosciences, respectively. Antibody against caspase 3 was
6
provided by Cell Signaling Technology. Antibodies against PARP, STAT3, HA-Tag, and
7
actin were acquired from Santa Cruz Biotechnology. CoCl2, DFO, 17-AAG, deguelin,
8
cisplatin, H2O2, 3-(4,5-dimethylthiasol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
9
Hoechst 33342, and actinomycin D were acquired from Sigma-Aldrich. ER tracker Blue-
10
White DPX and 2′-7′-dichlorofluorescin diacetate (DCF-DA) were purchased from
11
Invitrogen. [γ-32P]ATP was purchased from Amersham Biosciences.
12
13
RNA interference
14
siRNAs specific to CypB and scrambled control siRNA were purchased from
15
Dharmacon (Thermo Scientific), and siRNAs specific to HIF-1α were obtained from Santa
16
Cruz Biotechnology. The siRNAs (100 nM) were transfected into cells by using
17
Lipofectamine 2000 (Invitrogen). The efficiency of siRNA interference of CypB and HIF-
1
1
1α was monitored by Western blot analysis.
2
3
Promoter analysis and luciferase assay
4
The CypB promoter sequence was analyzed by using Genomatix MatInspector
5
(http://www.genomatix.de). For construction of luciferase reporter plasmids, 1,000 bp of
6
the CypB promoter sequence was amplified through PCR. The amplified fragments were
7
cloned into the pGL3 basic vector (Promega) with KpnI and XhoI. For mutational analysis,
8
the HRE site was mutated by PCR-based site-directed mutagenesis. Primers used for
9
constructing luciferase reporters containing various regions of the CypB promoter or
forward,
5′-
pGL3-CypB-150
reverse,
5′-
AACTCGAGTAGGTCCCCCTGCGGGGCGG-3′;
pGL3-CypB-350
forward,
5′-
13
AAGGTACCCTGAAGTGTTTGGAAGCCAC-3′;
pGL3-CypB-350
reverse,
5′-
14
AACTCGAGTAGGTCCCCCTGCGGGGCGG-3′;
pGL3-CypB-800
forward,
5′-
15
CCGGTACCGGACAACACTAAGTGTGTGA-3′;
pGL3-CypB-800
reverse,
5′-
16
AACTCGAGTAGGTCCCCCTGCGGGGCGG-3′;
pGL3-CypB-150M
forward,
5′-
17
AGCCCGGGCCAAAGCTCTGCGA-3′;
10
mutated
11
AAGGTACCAGGCCCACGTATTTGCTAAC-3′;
12
HRE
were
as
follows:
pGL3-CypB-150
pGL3-CypB-150M
2
reverse,
5′-
1
TCGCAGAGCTTTGGCCCGGGCT-3′;
2
GACAAGCTTCCAAAGCCCCTTGTCC-3′;
3
GGACAAGGGGCTTTGGAAGCTTGTC-3′.
pGL3-CypB-350M
pGL3-CypB-350M
forward,
reverse,
5′5′-
4
Huh7 and HepG2 cells were transfected with 0.2 μg of the pGL3 basic vector-
5
derived plasmids together with the internal control plasmid, pCMV-Lac (Promega).
6
Luciferase and β-galactosidase (β-gal) activities (not shown) in 50 μl cell lysates were
7
measured in a microplate reader (Bio-Rad), and the luciferase activity was normalized to
8
the β-gal activity.
9
10
11
EMSA
EMSA
was
performed
with
oligonucleotides
CypB-M,
(CypB/WT,
5′5′-
12
GACAAGCTTCCCGTGCCCCTTGTC-3′;
13
GACAAGCTTCCAAAGCCCCTTGTC-3′) derived from the CypB promoter sequence.
14
Sense and antisense strands of the oligonucleotides were annealed into double-stranded
15
oligonucleotides and labeled with [γ-32P]ATP. Nuclear extracts were obtained from Huh7
16
cells before and after hypoxic incubation for 24 h. The nuclear extracts (10 μg) were
17
incubated in the presence of 5× binding buffer (25% glycerol, 50 mM Tris-HCl [pH 7.5],
3
1
250 mM NaCl, 5 mM dithiothreitol [DTT], 1 mg/ml poly(dI-dC), 5 mM
2
ethylenediaminetetraacetic acid [EDTA]). For a competition study, a 100-fold molar excess
3
of unlabeled oligonucleotides was added to the reaction mixture before the addition of the
4
radiolabeled probe. The samples were run on 5% nondenaturing polyacrylamide gels. The
5
gels were then dried and exposed to X-ray film with an intensifying screen at −80°C.
6
7
ChIP assay
8
Conventional ChIP was conducted as described previously.3 Cross-linked Huh7
9
and HepG2 chromatin was subjected to immunoprecipitation with antibodies against HIF-
10
1α,
CypB,
and
STAT3.
CypB-HRE
was
11
GGCGGTAAGGATAAATGTCCCTGAAG-3′
12
AACGAGAAGACCACAAGCCCCGGCAT-3′.
amplified
by
and
using
primers
5′5′-
13
14
Establishment of stable cell line
15
pcDNA3-CypB/WT (3 μg) or pcDNA3 (3 μg) alone was transfected into Huh7,
16
PLC/PRF/5, HepG2 and Hep3B cells by using Lipofectamine 2000 (Invitrogen). For stable
17
transfection, the cells were cultured in selective medium with 800 μg/ml G418 for a month.
18
Then, drug-resistant individual clones were isolated and incubated for further amplification
4
1
in the presence of selective medium.
2
3
MTT assay
4
MTT assay was conducted in 12-well plates. The optical density was assessed at
5
550 nm with a microplate reader (Bio-Rad). Cell survival was expressed as the percentage
6
of absorbance of the treated cells relative to that of the untreated cells.
7
8
VEGF assay
9
VEGF concentrations in Huh7 and HepG2 cells were quantified by using a VEGF enzyme-
10
linked immunosorbent assay (ELISA) kit (Ray Biotech, Inc.) according to the
11
manufacturer's instructions.
12
13
14
15
TUNEL assay
TUNEL assay was performed by using the ApopDIRECT DNA fragmentation kit
(Invitrogen). Positive apoptotic nuclei were assessed by confocal microscopy.
16
17
18
IHC analysis
Tissue microarrays (TMA) were designed and constructed using paraffin blocks
5
1
from HCC and colon cancer tissues. Paraffin sections were dewaxed using xylenes and
2
hydrated using a series of ethanol. Endogenous peroxidases were quenched with a short
3
treatment of 1% hydrogen peroxide. To enhance the signal, we used antigen retrieval in
4
citrate buffer and signal amplification with biotinylated tyramide. The deparaffinized and
5
rehydrated specimens were incubated overnight at 4°C with a monoclonal antibody against
6
CypB (1:14,000 dilution). The immunostained section was visualized with an EnVision
7
Detection Kit (Dako). Routine hematoxylin and eosin (HE)-stained sections were examined
8
to ensure the structural integrity of the tissues. The results were interpreted independently
9
by two pathologists who were blinded to the specific diagnosis and prognosis of each case.
10
The staining intensities were interpreted as negative when immunostaining was weak, and
11
positive when >30% of the cancer cells showed distinct immunoreactivity compared with
12
normal tissue (1-3). Immunoreactivity was assessed with a positive grading system as
13
described previously (4): +, minimal staining; ++, uniform or intense staining; +++,
14
intenser staining. Only the specimens exhibiting greater than + immunoreactivity were
15
considered positive.
16
17
Western blot analysis
6
1
Total cell extracts were separated by sodium dodecyl sulfate polyacrylamide gel
2
electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. After
3
blocking, the membrane was incubated with the indicated primary antibodies, followed by
4
incubation
5
chemiluminescence reagents (Santa Cruz Biotechnology).
with
secondary
antibody.
Samples
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
7
were
detected
with
enhanced
1
Supporting References
2
3
1. Tak E, Lee S, Lee J, Rashid MA, Kim YW, Park JH, Park WS, et al. Human carbonyl
4
reductase 1 upregulated by hypoxia renders resistance to apoptosis in hepatocellular
5
carcinoma cells. J Hepatol;54:328-339.
6
7
2. Yoon JH, Song JH, Zhang C, Jin M, Kang YH, Nam SW, Lee JY, et al. Inactivation of
the Gastrokine 1 gene in gastric adenomas and carcinomas. J Pathol;223:618-625.
8
3. Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, Dowsett M,
9
et al. American Society of Clinical Oncology/College of American Pathologists
10
guideline recommendations for human epidermal growth factor receptor 2 testing in
11
breast cancer. J Clin Oncol 2007;25:118-145.
12
4. Sosa MS, Lopez-Haber C, Yang C, Wang H, Lemmon MA, Busillo JM, Luo J, et al.
13
Identification of the Rac-GEF P-Rex1 as an essential mediator of ErbB signaling in
14
breast cancer. Mol Cell;40:877-892.
15
16
17
18
19
20
21
22
8
1
Supporting Information
2
3
Supporting Figure 1. (A) Flow cytometric analysis for ROS measurement in transfected
4
HepG2 cells exposed to hypoxic conditions for 48 h. (B) Western blot analysis of cleaved
5
PARP and cleaved caspase 3 in HepG2 cells transfected with pcDNA3-CypB/WT (left) or
6
CypB siRNA (right) and exposed to the different treatments. (C) Comet assay of
7
transfected HepG2 cells after cisplatin treatment. The arrowheads represent chromosomal
8
DNA fragmentation as a sign of cisplatin-induced apoptosis. Scale bar, 100 μm. (D)
9
TUNEL assay of transfected HepG2 cells under hypoxia. The arrows indicate TUNEL-
10
positive cells. Scale bar, 20 μm.
11
12
Supporting Figure 2. (A) Co-immunoprecipitation of CypB with STAT3 in HepG2 cells
13
treated without or with hypoxia for 12 h and then analyzed with CypB or STAT3 antibody.
14
Western blot analysis was conducted with the antibodies by using cell lysates (input). (B)
15
Redistribution of CypB in HepG2 cells exposed to normoxic or hypoxic conditions for 12 h
16
followed by immunostaining with CypB antibody and FITC secondary antibody. Scale bar,
17
20 μm. (C) ChIP assay of the STAT3-binding site within the −209 bp region in HepG2 cells
18
exposed to hypoxia for 12 h (H) or unexposed (N) and then analyzed with CypB or STAT3
9
1
antibody. Input, amplified HIF-1α from a 1:100 dilution of total input chromatin as positive
2
control; IgG, immunoprecipitation with nonspecific IgG as negative control. (D) Luciferase
3
reporter assay of HepG2 cells transiently transfected with CypB siRNA and luciferase
4
reporter gene plasmids pGL3-VEGF-HRE-Luc, pGL3-EPO-HRE-Luc, and pGL3-GLUT1-
5
HRE-Luc or β-gal expression vector pCMV, and then treated for an additional 12 h under
6
hypoxic conditions. The data are the mean ± SEM from six independent experiments. *P <
7
0.05 compared with scrambled siRNA in hypoxia.
8
9
Supporting Figure 3. (A) Hypoxia-induced VEGF production regulated by CypB. VEGF
10
was analyzed in media collected from HepG2 cells using an ELISA kit, and its
11
concentration was calculated according to the curve plotted by standard VEGF. The data are
12
the mean ± SEM from six independent experiments. *P < 0.05 compared with Mock in
13
hypoxia, #P < 0.05 compared with scrambled siRNA in hypoxia. (B) Endothelial tube
14
formation. HepG2 cells were transfected with CypB/WT or CypB specific-siRNA, and
15
incubated under normoxia or hypoxia in serum free medium for 24 h to collect conditioned
16
medium. Human umbilical vein endothelial cells were incubated in medium containing
17
conditioned medium on Matrigel coated plates. The data are the mean ± SEM. *P < 0.05
10
1
compared with Mock in hypoxia, #P < 0.05 compared with scrambled siRNA in hypoxia.
2
Scale bar, 200 μm.
3
11