SUPPLEMENTARY INFORMATION
Supplementary Materials and Methods
RNA extraction, reverse transcription (RT) and real-time PCR. Total RNA from
cultured cells was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA) as the
manufacturer instructed. cDNAs were amplified and quantified in ABI Prism 7500 Sequence
Detection System (Applied Biosystems, Foster City, CA) using dye SYBR Green I
(Molecular Probes, Invitrogen, Carlsbad, CA). Expression data were normalized to the
geometric mean of housekeeping gene GAPDH to control the variability in expression levels
and calculated as 2-[(C t of gene) – (C t of GAPDH)], where Ct represents the threshold cycle for each
transcript.
Primers and Oligonucleotides. Real-time PCR primer: AKIP1, forward: 5’-CCAACCCTT
AGTGCTTCC-3’ and reverse: 5’-TCGACTCGCCTCTGTGATA-3’; VEGF-C, forward: 5’-C
GGACTCGACCTCTCGG-3’ and reverse: 5’-TGGACACAGACCGTAACTGC-3’; GAPDH:
5’-ATTCCACCCATGGCAAATTC-3’ and 5’-TGGGATTTCCATTGATGACAA G-3’.
Primers used for human VEGFC promoter reporter construction: P1-luc, forward: 5’-GCC
CCGCGGGAATCCTGATGTCCAAGCCT-3’ and reverse: 5’-GCCCTGCAGGTTCTCCG
CAAACCCTAACG-3’; P2-luc, forward: 5’-GCCCCGCGGCTCCAGGAG TCAAAAGCA
ATAG-3’; P3-luc, forward: 5’- GCCCCGCGGGGATAAAGCGGGGG CTCCC-3’; P4-luc,
forward: 5’- GCCCCGCGGGCGTCAGTCATGCCCTGC-3’; P5-luc, reverse: 5’- GCCCTG
CAGTGAGCAACACTGCTGAAAAG-3’; P6-luc, forward: 5’- GCCCCGCGGCTCCAGG
AGTCAAAAGCAATAG-3’ and reverse: 5’- GCCCTGCAGCCTCCAGAGCGCGCCGGG
CT-3’.
VEGFC promoter primer used for ChIP assays: F1, forward: 5’-CCACCTTCTATTC
AAATCCTAC-3’ and reverse: 5’- TGCTGTCTTCCTGCTCTATG-3’; F2, forward: 5’-
TTAGACGTGACTAAGTAGCAG-3’ and reverse: 5’- ATTGAGTTTATTTTCTGGCA-3’;
F3, forward: 5’-GGCTGCCAGAAAATAAACT-3’ and reverse: 5’-ATGAAGTATGCGGTT
GAGAA-3’; F4, forward: 5’-CTGTCATCTTGGGGAATAAA-3’ and reverse: 5’-CTCTATT
GC TTTTGACTCCTG-3’; F5, forward: 5’-CAGTGTTGCTCATCAATAAAGG-3’ and
reverse: 5’-ATTTGAAACTCCTCACCCAT-3’. F6, forward: 5’-AAGAAAGTCTCTTCTTC
CGG-3’ and reverse: 5’-CAGGGTGAGCAGGTTACAG-3’; F7, forward: 5’-GCAGTCACT
TTTCTT TGGAT-3’ and reverse: 5’-CTTCTGCAAGAAGTTGGTTT-3’; F8, forward: 5’-A
CTGCAT CCTGAGAACTGC-3’ and reverse: 5’-CAAAGTTTGGAAATGTCCTG-3’; F9,
forward: 5’-GTGGTCGCATCACCTCTAA-3’ and reverse: 5’-CTCCCCTCTCACTCTCCC
T-3’; F10, forward: 5’-TCTGGCGGGTTTGGAGGG-3’ and reverse: 5’-TGAAGCGAGGG
CGAGGGA -3’; F11, forward: 5’-GTTTCCTGTGAGGCTTTTACC-3’ and reverse: 5’-CTC
CTGGTCCCTCTCCCC-3’.
AKIP1 scramble: GGUAGCCAUUACACCAUAAGU;
AKIP1 RNAi#1: CCUUCAGA
ACAAUGGCUGAAU (targets coding sequences within AKIP1 exon 3);
AKIP1 RNAi#2:
GUCAGUGUGGGAAAUAUUAUUCA (targets coding sequences within AKIP1 exon 3);
VEGFC scramble: GGCAAAUAACACUAUAUACCU;
VEGFC siRNA#1: CAAACCAG
UAACAAUCAGUUU; VEGFC siRNA#2: CCACCAAACAUGCAGCUGUUA.
scramble (Scr-1): GAGGGAGGUUGCGUGUAAU;
GGAAUA;
AAACA;
SP1 siRNA: GGCUGGUGGUGAU
AP-2 scramble (Scr-2): GGACCCACUAAUAGAUUGU;
GGGUAUUAACAUCCCAGAU;
AP-2 siRNA:
NF-B p65 scramble (Scr-3): GACCGCGACAACGU
NF-B p65 siRNA: GGAGCACAGAUACCACCAA;
UUCCGGCAAUCUAU;
PKA scramble: GUACC
PKA siRNA: UCCUGCAAGCUGUCAACUU;
GCGACGUUAGGUGCAGUAU;
SP1
SirT1 scramble:
SirT1 siRNA: GCAGAUUAGUAGGCGGCUU;
Dorsal air sac assay. The dorsal air sac assay was used to examine the angiogenic response
triggered by cells. Briefly, the chamber consisting by a ring (Millipore, Billerica, MA)
covered with filters (0.45 μm pore size) at both sides was filled with 1×106 cells suspended
in 150 μl of PBS, and then implanted into a air sac in the dorsum of anesthetized male nude
mice (6 weeks old). On day 7, the chambers were removed from the subcutaneous fascia of
the mice, and the angiogenic response was assessed by determining the areas of new blood
vessels under a stereomicroscope.
Chemical reagent. NF-B inhibitor, Helenalin, was purchased from EMD biosciences
(Gibbstown, NJ) and dissolved in Dimethyl sulfoxide (DMSO). DMSO was used
throughout the experiments as the vehicle control.
Chicken chorioallantoic membrane (CAM) assay.
CAM assay was performed at the 8th
day of development of fertilized chicken eggs according to a method described previously.1 A
1-cm diameter window was opened in the shell of each egg with 8-day-old chicken embryo
(Yueqin Breeding Co. Ltd, Guangdong, China).
The surface of the dermic sheet on the
floor of the air sac was removed to expose the CAM. A 0.5-cm diameter filter paper was
first placed on top the CAM, and 100μl conditioned media harvested from ESCC cells were
added onto the center of the paper. Then the eggs were incubated at 37°C under 80–90%
relative humidity for 48 hours. Following fixation with stationary solution (methanol:
acetone=1:1) for 15min, CAMs were cut and harvested, and gross photos of CAMs were
taken under a digital camera (Panasonic, Osaka, Japan). The effect of conditioned media
harvested from different cells was evaluated by the number of second- and third-order vessels
in comparison with that treated with the medium harvested from control group.
References
1. Célérier J, Cruz A, Lamandé N, Gasc JM, Corvol P. Angiotensinogen and its cleaved
derivatives inhibit angiogenesis. Hypertension. 2002; 39:224-228.
2. Cristea IM, Chait BT. Conjugation of magnetic beads for immunopurification of protein
complexes. Cold Spring Harb Protoc. 2011:pdb.prot5610.
Supplementary Figure Legends
Supplementary Figure 1. AKIP1 is overexpressed in ESCC cell lines and primary
human ESCC.
(a) Western blotting analysis revealed that AKIP1a, the longest isoform of
AKIP1, is predominately expressed in the Kyse30 ESCC cells, using overexpressed
HA-tagged AKIP1 splice variants as controls.
α-Tubulin was used as a loading control.
(b-c) Real time-PCR analysis of AKIP1 mRNA expression in 2 NEECs and 12 cultured
ESCC cell lines (b), and in 10 paired primary ESCC tissues (T) and adjacent non-cancerous
tissues (ANT) from the same patient (c). Expression levels were normalized to GAPDH.
(d) Quantification analysis of AKIP1 protein expression in 10 paired human ESCC tissues (T)
and the matched adjacent non-tumor tissues (ANT) from the same patient (as shown in
Figure 1b).
(e) Statistical analysis of the average MOD of AKIP1 staining for ESCC
specimens of different clinical stages. The average MOD of AKIP1 staining increases as
ESCC progresses to higher stages. Each bar represents the mean ± SD of three independent
experiments.
* P < 0.05.
Supplementary Figure 2.
(a-b) Kaplan-Meier curves and univariate log-rank analyses
of the survival of ESCC patients expressing low and high levels of AKIP1 within
subgroups of Clinical stage I (a) and T1 classification (b).
Samples with an IHC scoring
index (SI) ≥ 8 were determined as high expression and samples with a SI < 8 were
determined as low expression (For full details of the IHC SI see Supplemental Materials and
Methods).
Supplementary Figure 3.
The effect of dysregulation of AKIP1 on ESCC progression
examined using an in vivo tumor model.
(a) Western blotting analysis of AKIP1
expression in the Kyse30/AKIP1-RNAi tumors, the Kyse30/RNAi-vector tumors and control
tumors, respectively.
α-Tubulin was used as a loading control.
(b) Tumor volumes were
measured on the indicated days.
(c) Mean tumor weights.
* P < 0.05.
Supplementary Figure 4. AKIP1 enhances the ability of ESCC cells to induce
angiogenesis.
(a) Representative images (left panel) and quantification (right panel) of
TECs tube formation cultured on Matrigel-coated plates with conditioned media from vector
control cells and AKIP1-transduced cells, or RNAi-vector control cells and AKIP1-silenced
cells. The capillary tubes were quantified by counting length (10 random 100× fields per
well). (b) Cell migration assay was performed by culturing TECs with conditioned medium
derived from the indicated ESCC cells.
Migratory cells on the lower membrane surface
were fixed in 1% paraformaldehyde, stained with hematoxylin, and counted (10 random
100× fields per well). (c) Representative images (left panel) and quantification (right panel)
of the blood vessels formed in the CAM assay after stimulation with conditioned medium
from the indicated cells.
(d) Representative graphs of mouse dorsal air-sac angiogenesis
induced by vector cells and AKIP1-overexpressing cells, or RNAi-vector cells and
AKIP1-silenced cells.
Each bar represents the mean ± SD of three independent experiments.
Each bar represents the mean ± SD of three independent experiments.
* P < 0.05.
Supplementary Figure 5. AKIP1 promotes ESCC lymphangiogenesis. (a)
Representative images (left panel) and quantification (right panel) of LECs tube formation
cultured on Matrigel-coated plates with conditioned medium from vector control cells and
AKIP1-transduced cells, or RNAi-vector control cells and AKIP1-silenced cells.
(b) Cell
migration assay was performed by culturing LECs with conditioned media derived from the
indicated ESCC cells. Error bars represent the mean ± SD of three independent experiments.
* P < 0.05.
Supplementary Figure 6. AKIP1 upregulates VEGF-C expression.
(a-b) Real-time PCR
(A) and IHC (B) analyses of VEGF-C expression in the tumors formed by vector control
cells, AKIP1-transduced cells, RNAi-vector cells and AKIP1-silenced cells.
(c) Western
blotting analysis of the expression levels of phospho-AKT (Ser 473) and phospho-ERK1/2
(T202/Y204) in the TECs treated with conditioned medium from AKIP1-transduced Kyse30
cells and in the TECs treated with conditioned medium from AKIP1-silenced KYSE30 cells.
α-Tubulin was used as a loading control.
the indicated cells.
(d) Western blotting analysis of COX2 protein in
α-Tubulin was used as a loading control.
(e) AKIP1 transcriptionally
regulates the expression of VEGF-C through physically associating with the VEGF-C
promoter.
Transactivation activity of serial fragments of the VEGF-C promoter in vector-
or AKIP1-transduced KYSE510 cells. The cells were transfected with vectors expressing a
luciferase reporter gene driven by various indicated fragments of the VEGF-C promoter (as
shown in Figure 5b). The luciferase activities of the promoter constructs were measured
and normalized to Renilla luciferase activity.
(f) Three mutants of VEGF-C promoter were
constructed and luciferase acitivity assay was performed.
SD of three independent experiments.
Error bars represent the mean ±
* P < 0.05.
Supplementary Figure 7. AKIP1 regulates VEGF-C promoter activity in ESCC cells.
(a) Real time-PCR analysis of VEGF-C mRNA expression (left panel) and Western blotting
analysis of SP1, AP-2 and NF-κB p65 expression (right panel) in the indicated cells.
VEGF-C mRNA expression levels were normalized to GAPDH. α-Tubulin was used as a
protein loading control.
(b) ChIP analysis of the binding efficiency of AKIP1 for different
regions of VEGF-C promoter in the indicated cells treated with or without NF-
.
(c) Overexpressing AKIP1 increased, but silencing AKIP1 decreased, the binding efficiency
of SP1, AP-2 and NF-
the VEGF-C promoter.
Error bars represent the mean ± SD of
three independent experiments. * P < 0.05.
Supplementary Figure 8. Silencing AKIP1 decreases VEGF-C expression in NSCLC,
HCC and ovarian cancer cells.
Left panel: Real time-PCR analysis of VEGF-C mRNA
expression in A549, HepG2 and SK-OV-3 cells transfected with scrambled control or AKIP1
siRNA(s). Expression levels were normalized to GAPDH. Right panel: Western blotting
analysis of AKIP1 expression in the indicated cells.
control.
α-Tubulin was used as a loading
Each bar represents the mean ± SD of three independent experiments. * P < 0.05
Supplementary Tables
Supplementary Table 1. Clinicopathological characteristics of studied patients and
expression of AKIP1 in ESCC
Factor
NO.
(%)
235
78.1
66
21.9
≤57
152
50.5
>57
149
49.5
I
40
13.3
II
136
45.2
III
92
30.5
IV
33
11.0
T1
45
15.0
T2
71
23.6
T3
172
57.1
T4
13
4.3
N0
155
51.5
N1
146
48.5
No
268
89.0
Yes
33
11.0
90
29.9
Gender
Male
Female
Age (years)
Clinical stage
T classification
N classification
M classification
Histological Grade
Well
Moderate
120
39.9
91
30.2
Alive
98
32.6
Dead
203
67.4
Low expression
134
44.5
High expression
167
55.5
Low expression
124
41.2
High expression
177
58.8
Poor
Vital status
Expression of AKIP1
MVD (Expression of CD31)
Supplementary Table 2. Correlation between the clinicopathological features and
expression of AKIP1
AKIP1 expression
Patient characteristics
Low or none
High
Male
102
133
Female
32
34
≤57
68
84
0.463
Gender
0.939
Age (years)
>57
66
83
I
30
10
II
44
75
III
132
61
62
32
IV
11
22
T1
31
14
T
T2
44
40
classification
T3
132
31
62
67
105
T4
5
8
N
N0
89
66
classification
N1
204
45
0
101
M
No
123
145
classification
Yes
11
22
Well
48
42
Moderate
174
50
70
Poor
36
55
Alive
67
31
Dead
67
136
MVD
Low
83
41
expression
High
51
126
Clinical stage
Histological
P-value
< 0.001
60
0.004
< 0.001
0.171
Grade
0.127
< 0.001
Vital status
< 0.001
Supplementary Table 3. Univariate and multivariate analysis of different prognostic
parameters in patients with ESCC by Cox-regression analysis
Univariate analysis
Multivariate analysis
P
Hazard ratio
(95% CI)
P
Hazard ratio
(95% CI)
< 0.001
2.065 (1.564-2.725)
0.816
0.969 (0.744-1.262)
< 0.001
1.385 (1.165-1.645)
0.154
1.198 (0.935-1.535)
< 0.001
2.371 (1.788-3.144)
0.005
1.662 (1.169-2.362)
< 0.001
2.905 (2.154-3.917)
< 0.001
2.125 (1.529-2.952)
Clinical stage
I
II
III
IV
T classification
T1
T2
T3
T4
N classification
N0
N1
AKIP1 expression
Low expression
High expression