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Supplementary MATERIALS AND METHODS
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clinicopathological data, including sex, serum AFP level, hepatitis C virus (HCV),
hepatitis B surface antigen (i.e., HBV infection), and preoperative hepatic functional
reserve as determined by Child-Pugh classification.
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Cell culture
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Patients and Tumor Materials
The histological diagnosis of HCC was confirmed in all cases based on the latest
World Health Organization classification. Grading of histological differentiation was
assigned based on Edmonson and Steiner criteria, while tumor staging was
determined according to the American Joint Committee on Cancer staging system
(AJCC, 7th 8 edition), Okuda staging system, and the Cancer of the Liver Italian
Program (CLIP) score (Takanishi et al. Hawaii Med J 2007;66:209-12). Medical
charts were reviewed for each patient to ascertain the accuracy of other
Primary human hepatocytes (#5200, Sciencell Research Laboratories) were
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cultured with recommended medium (#5201, Sciencell) in poly-L-lysine (2 μg/cm2)
coated flasks. Hep-3B and Huh-7 HCC-derived cells were used to performed in vitro
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studies. Huh-7 is a well differentiated hepatocyte derived cellular carcinoma cell line
that was originally taken from a liver tumor in a 57-year-old Japanese male in 1982,
established by Nakabashi H and Sato J. Huh-7 cells express TP53, CDKN1A (also
known as p21Cip1), AFP, cytochrome P450 and albumin (Clayton et al. 2005, Liver
International 25:389-402; Hsieh et al. 2003, Clin Cancer Res. 9:338-345) and
HBV-negative (Japanese Collection of Research Bioresources; Cha et al. 2004;
Hepatology 39:1683-1693).
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Quantitative Reverse Transcription-Polymerase Chain Reaction, Synchronizing Cell
Cycle and Flow Cytometric Analysis
To analyze the relative mRNA expression levels of E2F1 and STMN1, absolute™
QPCR SYBR® Green Mix (ABgene) and iCycler (Bio-Rad Laboratories) were used,
followed by normalization to a housekeeping transcript, polymerase (RNA) II (DNA
directed) polypeptide A (POLR2A), and a common reference cDNA pooled from
equal aliquots of each sample. Primer sets POLR2A (4 µM; 127 bp):
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5’-CCCACTTACTCGCCCACTTCC-3’, 5’-TTCTCCTCGTCACTGTCATCCG-3’;
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STMN1 (4 µM; 108 bp): 5’-AGAGAACCGAGAGGCACAAATGG-3’,
5’-TCGTCAGCAGGGTCTTTGGATTC-3’; E2F1 (4 µM; 73 bp):
5’-AGATGGTTATGGTGATCAAAGCC-3’,
5’-ATCTGAAAGTTCTCCGAAGAGTCC-3’ and AFP (4 µM; 77 bp):
5’-GAGGGAGCGGCTGACATTATT-3’, 5’-TGGCCAACACCAGGGTTTA-3’
were used. Primer concentrations were optimized to achieve equivalent PCR
efficiencies among the control POLR2A and each target gene (Shiue et al.
Theriogenology 2006;66:1274-83). Reactions were carried out in triplicate with
annealing temperature of 60°C, 56°C, 60°C and 53°C for E2F1, STMN1, CCNA2 and
AFP versus POLR2A, respectively. The Coulter®Epics® XL™ flow cytometer
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(Beckman Coulter) and WinMDI version 2.9 software were used for cell cycle
analysis.
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Immunoblotting Analysis
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Calbiochem), E2F1 (1:100; #sc-251, KH95, Santa Cruz), MYC (1:100; #sc-764,
N-262, Santa Cruz), TP53 (1:200; #sc-126; DO-1, Santa Cruz), RB1 (1:200; #sc-102,
IF8, Santa Cruz) and TFDP1 (1:200; #sc53642, TFD10, Santa Cruz) were used as
primary antibodies. Protein bands were detected by Western Lightning™
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Chemiluminescence Reagent Plus Kit (Perkin-Elmer Life Sciences) with horseradish
peroxidase-labeled secondary antibody as suggested by the manufacturer and
visualized on a VersaDoc Imaging System (Bio-Rad).
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Cell lysates were prepared with RadioImmunoPrecipitation Assay buffer (Upstate
Biotechnology). Lysates containing equal amounts of protein were separated by
12-15% SDS-PAGE and electroblotted into polyvinylidene fluoride membrane
(Immobilon TM-P Transfer Membrane, Millipore Corporation). Antibodies against
actin, beta (ACTB; 1:3,000; #MAB1501, Chemicon), STMN1 (1:200, #569391,
Immunohistochemistry
Two pathologists (Huang HY and Li CF) blinded to clinicopathological data and
patient outcomes, independently interpreted immunostains of anti-STMN1 (1:20).
Immunohistochemical staining was carried out on representative tissue sections cut
from formalin-fixed, paraffin-embedded tissues at 5-lm thickness. Sections were
deparaffinized with xylene-free EZ-Dewax™ 50 solution (BioGenex Laboratories)
and rehydrated. Bond Epitope Retrieval Solution 1 was used to perform antigen
retrieval of STMN1 at 100°C for 20 min, respectively. For antigen retrieval of
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STMN1, Bond Epitope Retrieval Solution 2 was applied at 100°C for 20 min. All
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slides were washed using Bond™ Wash Solution, and immunostains were performed
using the Bond-max™ automated immunostainer (Vision BioSystems), following the
standard protocol F of the manufacturer’s instructions. After washing, slides were
incubated for 30-40 min at 25°C with primary antibodies. The Bond™ Polymer
Refine Detection Kit (Vision BioSystems) was used to detect immunoreactivity on
tissue sections, which were then counterstained with Gill’s hematoxylin. One breast
cancer specimen previously known to be positive for STMN1 was used as the positive
control and incubation without primary antibodies was used as a negative control.
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Transient Transfection
Cells at a density of 1.5 x 105 were seeded in six-well plates and transfected with
pCMV6-XL/TFDP1 (1 μg) and/or pCMV6-XL/E2F1 (1 μg) or pCMV6-XL plasmid
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(2 μg) (OriGene Technologies, Rockville, MD, USA) in 6 μL TransFast Transfection
reagent (Promega). After transfection, cells were incubated at room temperature for
20 min and transferred to an incubator with culture medium for 48 h before analysis.
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found to be effective. TRCN0000072243 (shLuc) was served as control. The shRNA
clones have been inserted into the pLK0.1 vector, downstream of the U6 promoter.
Heh-7 and Hep-3B cells at density of 3 x 106 were transfected with 2 μg shRNA
plasmids in 8 μL PolyJet™ DNA In Vitro Tranfection Reagent (SignaGen
Laboratories, Gaithersburg, MD, USA), according to the manufacturer’s instruction.
After transfection, cells were plated onto 6-cm culture dishes, maintained in a 37°C,
5% CO2 incubator for five days before analysis. Alternations in expression levels of
E2F1, STMN1 transcripts, E2F1 and STMN1 protein abundances were analyzed by
quantitative RT-PCR and immunoblotting analysis.
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Five shRNA clones targeting E2F1 genes (National RNAi Core Facility, Institute
of Molecular Biology, Academia Sinica, Taipei, Taiwan) were preliminarily screened,
only two (TRCN0000000250: shE2F1#3 and TRCN0000010328: shE2F1#5) were
Chromatin Immunoprecipitation and Quantitative Assays
Primers spanning -1,774 to -1,781 (Site 1) and -1,044 to -1,051 (Site 2) of STMN1
promoter region were 5’-AGGACTACACTTCCCGAGGTGCTTC-3’,
5’-TCTGGACCACACTCTGAGCACCAA-3’ and
5’-AGCTTGGGTGGCGGCAGGTT-3’,
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5’-TAACGGTCCAATCCGGGTAACTCC-3’. One non-specific primer pair, targeting
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outside of the STMN1 genomic region on chromosome 1,
5’-CCCTGGCCCTGTAGTCATTA-3’, 5’-CCTTGGTATGACCCCTTCAA-3’, was
severed as negative control.
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Cells were grown overnight in 100-mm dishes to ~60-70% confluency (5 x106),
cross-linked with formaldehyde, harvested, and subsequently sonicated to obtain
soluble chromatin. After dilution, the chromatin solutions were incubated with 5 μg
anti-E2F1 antibody (#sc-251, Santa Cruz, Biotechnology, Inc., CA) or 5 μg IgG pool
from rabbit serum (non-specific control), and satiated on a rotating platform at 4ºC
overnight. Immunocomplexes were recovered with preblocked protein A-Sepharose
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beads (Zymed Laboratories) at 65ºC for 4 h. Samples were next digested with
proteinase K (Sigma-Aldrich) for 1 h at 45ºC and the DNA from samples was
obtained by phenol/chloroform extraction and ethanol precipitation. Annealing
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temperature for PCR was 62ºC.
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ΔCt(anti-E2F1vector alone – inputvector alone) – ΔC(anti-IgGvector alone – inputvector alone).
Relative DNA abundance was calculated by 2-ΔΔCt for both test site and vector alone
and normalized to 2-ΔΔCt(vector alone). This experiment was triplicated and the
relative promoter occupancy was expressed as mean ± SEM.
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constructs were verified by sequencing. The mutagenic oligonucleotide primers
(mutations were underlined) were, Site 1: 5’CGTGCCTCTGTTTGGATCTTTTGTGCGCGCCC-3’, 5’GCACGGAGACAAACCTAGAAAACACGCGCGGG-3’ and Site 2:
5’-TGGAGGTGTTTTTGGATGGAGTTGGGGGGGGC-3’, 5’ACCTCCACAAAAACCTACCTCAACCCCCCCCG-3’.
Resultant PCR products were separated on 2%, 0.5 X Tris/borate/EDTA agarose
gels, stained with ethidium bromide; 94 visualized by UV light. For quantitative ChIP,
the relative copy number was determined using ΔΔCttest site = ΔCt(anti-E2F1E2F1/TFDP1
– inputE2F1/TFDP1) – ΔCt(anti-IgGE2F1/TFDP1 – inputE2F1/TFDP1) and ΔΔCtvector alone =
Generation of Reporter Constructs and Site-directed Mutagenesis
The single-mutant (Site 1: pGL3-A/m1E2F1) and double-mutant (Site 1 and Site 2:
pGL3-A/dmE2F1) were performed with the QuickChange Lightning Site-Directed
Mutagenesis Kit (#210518, Agilent), following the manufacturer’s instructions. All