Supplementary data 2:
Real-Time PCR
For real-time PCR, total RNA was converted to cDNA using SuperScript™ III First-Strand
Synthesis SuperMix for qRT-PCR (Invitrogen, CA) as per manufacture’s instructions. The
ABI 7700® real-time PCR system (Applied Biosystems, USA) was used for analysis. Multiple
gene markers distributed around the genome and three housekeeping genes were used for realtime PCR analysis using the SYBR® GreenER™ qPCR SuperMix for ABI PRISM®
(Invitrogen, CA). The sequence information of all the primers was listed in Table S2. 5 ng
total cDNA from both test genes and endogenous control genes was added to a 20 µl reaction
containing SYBR® GreenER™ qPCR SuperMix for ABI PRISM®, and 200 nM each of
primers. Triplicate reactions were performed for each marker in a 384-well plate using a twostep amplification program of initial denaturation at 95°C for 10 min, followed by 40 cycles
of 95°C for 20s and 60°C for 30s. A melting curve analysis step was carried out at the end of
the amplification, consisting of denaturation at 95°C for 1 min and re-annealing at 55°C for 1
min. Standard curves were generated from each experimental plate using serial 5-fold
dilutions of untreated cDNA. The geometric mean of Ct-value for each reaction was
calculated. Amplification efficiencies were calculated according to the equation E = 10(–1/slope)
previously described (Heid et al., 1996) and ranged from 90–104% for all gene markers; no
nonspecific amplification or primer dimer was observed in any of the reactions as confirmed
by the melt curve analysis. To compensate for potential differences in between markers, the
relative expressions was computed, based on the efficiency (E), normalized by a panel of
housekeeping genes, β-actin, HPRT, and GAPDH (e.g. relative expression = 2
– (Ct,Sample -
Ct,HKG) - (Ct,Control - Ct,HKG)
sample-control)
) and the Ct difference () of sample versus control (Ct
(e.g. relative expression = 2 – ddCt).
S2-1
Table S2: The sequences of the oligonucleotide primers used for real-time PCR.
Genes
Abbr.
Primer Sequence (5'-3')
v-akt murine thymoma viral
oncogene homolog 1
AKT1
Forward:
GCACAAACGAGGGGAGTACAT
Reverse:
CCTCACGTTGGTCCACATC
Ataxia telangiectasia
mutated
ATM
Forward:
BCL2-antagonist of cell
death
BAD
Forward:
TGGATCCAGCTATTTGGTTTGA
CCAAGTATGTAACCAACAATAGAAGAAGT
AG
CCCAGAGTTTGAGCCGAGTG
Reverse:
CCCATCCCTTCGTCGTCCT
Forward:
GGGTGGTTGGGTGAGACTC
Reverse:
AGACACGTAAGGAAAACGCATTA
Forward:
TCCGCATCAGGAAGGCTAGA
Reverse:
AGGACCAGGCCTCCAAGCT
Forward:
ATGGCAGCAGTAAAGCAAGC
Reverse:
CGGAAGAGTTCATTCACTACCTGT
Forward:
TTCCTGGGCATGGAGTC
Reverse:
CAGGTCTTTGCGGATGTC
Forward:
CATGCTGAAACTTCTCAACCAGAA
Reverse:
TGTAGGCTCCTTTTGGTTATATCATTC
Forward:
AGTGACAGGTATGGGCGTTCG
Reverse:
GCATCTATCCCCCCTAAAGTGG
Forward:
ACGAAGGTCTGCGCGTGTT
Reverse:
CCGCTGGCCATGAACTACCT
Forward:
TAAGACCTGTATCTCGTGGCTG
Reverse:
CCCTGGTTCACTGCTATCTCT
Forward:
GCTAGCAGACTTTGGACTAGCCAG
Reverse:
AGCTCGGTACCACAGGGTCA
Forward:
CTGGTGTTTGAGCATGTAGACC
Reverse:
AAACTGGCGCATCAGATCCTT
Forward:
CCTCATCCCGTGTTCTCCTTT
Reverse:
GTACCACCCAGCGGACAAGT
Forward:
CAACGCACCGAATAGTTACGG
Reverse:
AACTTCGTCCTCCAGAGTCGC
Forward:
GTGGAGACCCACCTGCTCA
Reverse:
GGACACATCAGATTTATCCAAATCC
Forward:
AGTGGTGGGGAATAAACGCC
Reverse:
TCTGGCTTTAAGTCACGGTGTA
Forward:
ATCAGCACTTTCTTGAGCAACA
Reverse:
TTGTGCCAAGTAAAAGGTCTCC
Forward:
AGATGGTTATGGTGATCAAAGCC
Reverse:
ATCTGAAAGTTCTCCGAAGAGTCC
Forward:
ACTGGCCCTCATCCACCATA
Reverse:
GGTTGGCAGTGTGGAGCAG
Forward:
CCCCTGTGGCTAACAGTTACA
Reverse:
AGGTAGCTTTTAAGGCTTGACTC
Forward:
TGCCTCTCCTCAATGACCCTGA
Reverse:
ATAGGTCCATGTCTGGCACGGA
Forward:
TGCACCACCAACTGCTTAGC
Reverse:
GGCATGGACTGTGGTCATGAG
Forward:
TGACACTGGCAAAACAATGCA
Reverse:
GGTCCTTTTCACCAGCAAGCT
Forward:
CGGAGGGATTTGTTCGTGTTG
Reverse:
AGCTCCTTTAGAGGATAGCAAGT
Forward:
CTCCAAGTGCCGAAAAAGGAAG
Reverse:
CACCTGTTCCCTGAGCATGTTG
Forward:
CAATGGCGGTGTGGTGTTC
Reverse:
AGCTCCCTTATGATCTGGTTCC
BCL2-associated X protein
B-cell CLL/lymphoma 2
BCL2-like 1
Beta-actin
Reverse:
BAX
BCL2
BCL2L1
β-actin
Breast cancer 1, early
onset
BRCA1
Caspase 7, apoptosisrelated cysteine peptidase
CASP7
Cyclin D1
Cell division cycle 25A
Cyclin-dependent kinase 2
Cyclin-dependent kinase 4
Cyclin-dependent kinase
inhibitor 1A (p21, Cip1)
Cyclin-dependent kinase
inhibitor 2A (melanoma,
p16, inhibits CDK4)
CASP8 and FADD-like
apoptosis regulator
CHK2 checkpoint homolog
Cyclin E1
E2F transcription factor 1
CCND1
CDC25A
CDK2
CDK4
CDKN1A
CDKN2A
CFLAR
CHEK2
CCNE1
E2F1
v-erb-b2 erythroblastic
leukemia viral oncogene
homolog 2
v-ets erythroblastosis virus
E26 oncogene homolog 2
v-fos FBJ murine
osteosarcoma viral
oncogene homolog
Glyceraldehyde-3phosphate dehydrogenase
ERBB2
Hypoxanthine
phosphoribosyltransferase
HPRT
HIV-1 Tat interactive
protein 2, 30kDa
HTATIP2
v-jun sarcoma virus 17
oncogene homolog
JUN
Mitogen-activated protein
kinase kinase 1
MAP2K1
ETS2
FOS
GAPDH
S2-2
Amplicon
size (bp)
113
82
249
199
113
149
84
81
274
323
131
85
102
97
96
96
117
122
72
104
222
162
87
94
104
118
114
MET proto-oncogene
(hepatocyte growth factor
receptor)
Matrix metallopeptidase 1
Matrix metallopeptidase 2
Matrix metallopeptidase 9
Metastasis associated 1
Metastasis associated 2
MET
MMP1
MMP2
MMP9
MTA1
MTA2
v-myc myelocytomatosis
viral oncogene homolog
(avian)
Nuclear factor of kappa
light polypeptide gene
enhancer in B-cells 1
(p105)
Nuclear factor of kappa
light polypeptide gene
enhancer in B-cells
inhibitor, alpha
Non-metastatic cells 1,
protein (NM23A)
Phosphoinositide-3-kinase,
regulatory subunit 1 (p85
alpha)
Plasminogen activator,
urokinase
MYC
Plasminogen activator,
urokinase receptor
PLAUR
v-raf-1 murine leukemia
viral oncogene homolog 1
RAF1
Retinoblastoma 1
NFKB1
NFKBIA
NME1
PIK3R1
PLAU
RB1
S100 calcium binding
protein A4
Serpin peptidase inhibitor,
clade B (ovalbumin),
member 5
Serpin peptidase inhibitor,
clade E (nexin,
plasminogen activator
inhibitor type 1)
Synuclein, gamma (breast
cancer-specific protein 1)
S100A4
Telomerase reverse
transcriptase
Tumor necrosis factor
receptor superfamily,
member 10b
Tumor necrosis factor
receptor superfamily,
member 1A
Tumor necrosis factor
receptor superfamily,
member 25
Tumor protein p53 (LiFraumeni syndrome)
TERT
SERPINB5
SERPINE1
SNCG
TNFRSF10B
TNFRSF1A
TNFRSF25
TP53
Forward:
TGGTGCAGAGGAGCAATGG
Reverse:
CATTCTGGATGGGTGTTTCCG
Forward:
AGCTAGCTCAGGATGACATTGATG
Reverse:
GCCGATGGGCTGGACAG
Forward:
CAAAAACAAGAAGACATACATCTT
Reverse:
GCTTCCAAACTTCACGCTC
Forward:
TGGGGGGCAACTCGGC
Reverse:
GGAATGATCTAAGCCCAG
Forward:
GCTGTTACACCACACAGTCTT
Reverse:
GGACTCATGTTACTGCGGTTT
Forward:
CCGACGGCCTTATGCTCCT
Reverse:
CTGGGCCACCAGATCTTTGAC
Forward:
TGCTGCCAAGAGGGTCAAGT
Reverse:
GTGTGTTCGCCTCTTGACATTC
Forward:
TGCCAACAGATGGCCCATAC
Reverse:
TGTTCTTTTCACTAGAGGCACCA
Forward:
CTCCGAGACTTTCGAGGAAATAC
Reverse:
GCCATTGTAGTTGGTAGCCTTCA
Forward:
CTGCAGCCGGAGTTCAAAC
Reverse:
GCAATGAAGGTACGCTCACAGT
Forward:
GATTCTCAGCAGCCAGCTCTGAT
Reverse:
GCAGGCTGTCGTTCATTCCAT
Forward:
CACGCAAGGGGAGATGAA
Reverse:
ACAGCATTTTGGTGGTGACTT
Forward:
AATGGCCGCCAGTGTTACAG
Reverse:
CAGGAGACATCAATGTGGTTC
Forward:
TTTCCTGGATCATGTTCCCCT
Reverse:
ACTTTGGTGCTACAGTGCTCA
Forward:
GAACATCGAATCATGGAATCCCT
Reverse:
AGAGGACAAGCAGATTCAAGGTGAT
Forward:
GATGAGCAACTTGGACAGCAA
Reverse:
CTGGGCTGCTTATCTGGGAAG
Forward:
CTACTTTGTTGGCAAGTGGATGAA
Reverse:
ACTGGTTTGGTGTCTGTCTTGTTG
Forward:
CACAAATCAGACGGCAGCACT
Reverse:
CATCGGGCGTGGTGAACTC
Forward:
TGAGCAGCGTCAACACTGTG
Reverse:
GAGGTGACCGCGATGTTCTC
Forward:
GGAGCAAGTTGCAAAGCATTG
Reverse:
TCCCACGACGTAGTCCATGTT
Forward:
AAGACCCTTGTGCTCGTTGT
Reverse:
AGGTGGACACAATCCCTCTG
Forward:
TGCCTACCCCAGATTGAGAA
Reverse:
ATTTCCCACAAACAATGGAGTAG
Forward:
ACTGCCAACCATGCCTAGACTG
Reverse:
GAGCCTCCATCCCAGCTTC
Forward:
TGCAGCTGTGGGTTGATTCC
Reverse:
AAACACGCACCTCAAAGCTGTTC
S2-3
111
74
232
224
166
145
118
123
135
68
91
341
227
153
116
123
90
85
64
182
144
169
155
396
Table S3: Expression of genes related to oncogenic function in MCF7-ARTN relative to MCF7Vec cells.
Functional Gene
Grouping
Cell Cycle Control
& DNA Damage
Repair
Apoptosis and
Cell Senescence
Signal
Transduction
Molecules and
Transcription
Factors
Invasion and
Metastasis
Gene
Cyclin D1
ATM
BRCA1
Cyclin E1
CDC25A
CDK2
CDK4
CDKN1A
CDKN2A
CHEK2
E2F1
CDKN1B
RB1
S100A4
TP53
APAF1
BAD
BAX
BCL2
BCL2L1
CFLAR
CASP7
HTATIP2
TERT
TNFRSF1A
TNFRSF10B
TNFRSF25
AKT1
ERBB2
ETS2
FOS
JUN
MAP2K1
MYC
NFKB1
NFKBIA
PIK3R1
RAF1
SNCG
MET
MMP1
MMP2
MMP9
MTA1
MTA2
NME1
PLAU
PLAUR
SERPINB5
SERPINE1
Fold change#
p-value
-1.03
-1.27
-1.37
-1.04
-1.11
-1.13
-1.14
1.08
-2.76
-1.01
-1.05
-1.07
-1.00
1.27
0.00
1.13
1.03
-1.75
8.40
-1.22
-1.35
1.04
1.02
2.52
-1.12
-1.27
-1.25
-1.24
-1.03
-1.49
-1.70
-1.36
-1.12
1.01
-1.41
-1.03
-1.04
-1.22
1.67
-1.22
7.60
-1.32
1.29
-2.27
-1.41
-2.12
3.81
-1.04
1.29
3.10
3.21E-03
2.26E-02
8.99E-03
4.00E-03
2.66E-02
2.70E-05
2.70E-04
2.18E-05
2.19E-02
7.13E-03
5.04E-02
3.19E-04
1.92E-03
2.41E-01
2.85E-05
1.53E-02
4.83E-03
2.35E-02
1.05E-02
4.59E-02
2.76E-02
4.20E-04
1.13E-03
1.18E-02
1.71E-02
3.30E-02
1.19E-02
2.17E-04
3.62E-03
7.53E-03
5.61E-04
3.22E-04
8.32E-04
3.83E-04
6.03E-04
1.71E-02
2.58E-03
2.29E-05
4.99E-01
1.18E-03
2.64E-05
2.33E-02
1.49E-01
1.29E-03
3.12E-02
1.51E-02
3.39E-02
1.40E-02
4.54E-02
3.24E-03
#
Average of three experiments represents each gene fold-changes (p<0.05). A positive value
indicates increased gene expression and a negative value indicates decreased gene expression in
MCF7-ARTN cells relative to MCF7-Vec cells.
References
Heid CA, Stevens J, Livak KJ, and Williams PM. (1996). Real time quantitative PCR.
Genome Res 6: 986-994.
S2-4