10 A 0 C cg26821579 22851416 TSS200 cg26918510 22851422 ... · in BHY and Ca-Ski cell lines upon...
13
Tissue TargetID MAPINFO Position Ca-Ski HeLa SW756 COLO-680N KYSE-180 OACM 5.1C cg04255070 22851591 TSS1500 0.896 0.070 0.082 0.060 0.613 0.622 0.075 0.020 cg01593340 22851587 TSS1500 0.826 0.055 0.051 0.110 0.518 0.518 0.034 0.040 cg16113692 22851502 TSS200 0.964 0.073 0.072 0.030 0.625 0.785 0.018 0.010 cg26918510 22851422 TSS200 0.985 0.019 0.000 0.000 0.711 0.961 0.010 0.060 cg26821579 22851416 TSS200 0.988 0.083 0.058 0.000 0.387 0.955 0.021 0.050 cg01129966 22851401 TSS200 0.933 0.039 0.077 0.020 0.815 0.963 0.078 0.010 cg25421615 22851383 TSS200 0.958 0.033 0.006 0.000 0.447 0.971 0.037 0.020 cg17953636 22851321 1stExon 0.816 0.092 0.099 0.080 0.889 0.750 0.136 0.030 Cervix Normal cervical tissue Normal esophageal tissue Esophagus A D E B Supplemental Figure 1. Transcriptional silencing of SVIP by promoter CpG island hypermethylation in cervical and esophageal cancer cells. (A) SVIP expression analysis in head & neck, esophagus, cervix and haematological cell lines. A non-parametric Mann Whitney test was performed. (B) Bisulfite genomic sequencing of SVIP promoter CpG island in the studied cervical and esophageal cancer cells lines. CpG dinucleotides are represented as short vertical lines and the transcription start site (TSS) is represented as a long black arrow. Single clones are shown for each sample. Presence of an unmethylated or methylated cytosine is indicated by a white or black square, respectively. (C) DNA methylation profile of the CpG island promoter for the SVIP gene analyzed by the 450K DNA methylation microarray. Single CpG absolute methylation levels (0 – 1) are shown. Green, unmethylated; red, methylated. Data from three cervical (Ca-Ski, HeLa and SW756), three esophageal (COLO-680N, KYSE-180 and OACM 5.1C), normal cervical tissue and normal esophageal tissue are shown. (D) SVIP expression levels in cervical and esophageal cancer cell lines determined by real-time PCR (data shown represent mean.of biological replicates) (left) and western blot (right). (E) Expression of the SVIP RNA transcript (data shown represent the mean of biological replicates) (left) and protein (right) was restored in the SVIP epigenetically silent Ca-Ski, COLO-860N and KYSE-180 cells by treatment with the demethylating drug 5-aza-2’-deoxycytidine (AZA). qPCRs were analysed using a two tailed Student ‘s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. -SVIP -ACTB -SVIP -ACTB AZA - + - + - + ki a 6 0 N C 0.0 0.5 1.0 1.5 2.0 2.5 3.0 * ** *** *** mRNA expression (a.u.) Cervix Esophagus C mRNA expression (a.u.) i a 0 a N a 0.0 0.5 1.0 1.5 2.0 2.5 3.0 * ** * WT AZA-Treated -4 -2 0 2 4 * *** * *** SVIP mRNA expression (a.u.) Unmethylated Methylated -244 bp +104 bp TSS 244 bp +104 bp TSS 244 bp +104 bp TSS 244 bp +104 bp TSS 244 bp +104 bp TSS 244 bp +104 bp TSS Ca-Ski HeLa SW756 COLO-680N KYSE-180 OACM5.1 C - - - - -
Transcript of 10 A 0 C cg26821579 22851416 TSS200 cg26918510 22851422 ... · in BHY and Ca-Ski cell lines upon...
Tissue
TargetID MAPINFO Position Ca-Ski HeLa SW756 COLO-680N KYSE-180 OACM 5.1C
cg04255070 22851591 TSS1500 0.896 0.070 0.082 0.060 0.613 0.622 0.075 0.020
cg01593340 22851587 TSS1500 0.826 0.055 0.051 0.110 0.518 0.518 0.034 0.040
cg16113692 22851502 TSS200 0.964 0.073 0.072 0.030 0.625 0.785 0.018 0.010
cg26918510 22851422 TSS200 0.985 0.019 0.000 0.000 0.711 0.961 0.010 0.060
cg26821579 22851416 TSS200 0.988 0.083 0.058 0.000 0.387 0.955 0.021 0.050
cg01129966 22851401 TSS200 0.933 0.039 0.077 0.020 0.815 0.963 0.078 0.010
cg25421615 22851383 TSS200 0.958 0.033 0.006 0.000 0.447 0.971 0.037 0.020
cg17953636 22851321 1stExon 0.816 0.092 0.099 0.080 0.889 0.750 0.136 0.030
CervixNormal
cervical
tissue
Normal
esophageal
tissue
Esophagus
A
D
E
B
Supplemental Figure 1. Transcriptional silencing of SVIP by promoter CpG island hypermethylation in cervical and esophageal cancer cells. (A) SVIP expression analysis in head & neck,
esophagus, cervix and haematological cell lines. A non-parametric Mann Whitney test was performed. (B) Bisulfite genomic sequencing of SVIP promoter CpG island in the studied cervical and
esophageal cancer cells lines. CpG dinucleotides are represented as short vertical lines and the transcription start site (TSS) is represented as a long black arrow. Single clones are shown for each
sample. Presence of an unmethylated or methylated cytosine is indicated by a white or black square, respectively. (C) DNA methylation profile of the CpG island promoter for the SVIP gene analyzed
by the 450K DNA methylation microarray. Single CpG absolute methylation levels (0 – 1) are shown. Green, unmethylated; red, methylated. Data from three cervical (Ca-Ski, HeLa and SW756),
three esophageal (COLO-680N, KYSE-180 and OACM 5.1C), normal cervical tissue and normal esophageal tissue are shown. (D) SVIP expression levels in cervical and esophageal cancer cell
lines determined by real-time PCR (data shown represent mean.of biological replicates) (left) and western blot (right). (E) Expression of the SVIP RNA transcript (data shown represent the mean of
biological replicates) (left) and protein (right) was restored in the SVIP epigenetically silent Ca-Ski, COLO-860N and KYSE-180 cells by treatment with the demethylating drug 5-aza-2’-deoxycytidine
(AZA). qPCRs were analysed using a two tailed Student ‘s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
-SVIP
-ACTB
-SVIP
-ACTB
AZA - + - + - +
Ca-
Ski
HeL
a
SW
756
KYSE-1
80
COLO
-680
N
OACM
5.1
C
0.0
0.5
1.0
1.5
2.0
2.5
3.0
***
******
mR
NA
expre
ssio
n (
a.u
.)
Cervix Esophagus
C
mR
NA
expre
ssio
n (
a.u
.)
Ca-
Ski
Ca-
Ski A
za
KYS
E-1
80
KYS
E-1
80 A
za
COLO
-680
N
COLO
-680
N A
za
0.0
0.5
1.0
1.5
2.0
2.5
3.0
*
**
*
SV
IP r
ela
tiv
e e
xp
r (a
.u.)
BB30
-HNC
BHY
0.0
0.5
1.0
1.5
2.0
***
WT
AZA-Treated
*
HNC U
nmet
h
HNC M
eth
Eso
phagus
Unm
eth
Eso
phagus
Met
h
Cer
vix
Unm
eth
Cer
vix
Met
h
Hae
mat
ologic
al U
nmet
h
Hae
mat
ologic
al M
eth
-4
-2
0
2
4 * *** * ***S
VIP
rela
tive e
xp
ressio
n (
a.u
.)S
VIP
mR
NA
expre
ssio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
0
2
4
6
8
10
ns
Unmethylated
Methylated
TR
MT
12 m
RN
A E
xp
ressio
n
(lo
g2)
Ca-Ski
-244 bp +104 bp
TSS
244 bp +104 bp
TSS
244 bp +104 bp
TSS
244 bp +104 bp
TSS
244 bp +104 bp
TSS
244 bp +104 bp
TSS
HeLa
SW756
COLO-680N
KYSE-180
OACM 5.1C
Ca-Ski
-244 bp +104 bp
TSS
244 bp +104 bp
TSS
244 bp +104 bp
TSS
244 bp +104 bp
TSS
244 bp +104 bp
TSS
244 bp +104 bp
TSS
HeLa
SW756
COLO-680N
KYSE-180
OACM 5.1C
Ca-Ski
HeLa
SW756
COLO-680N
KYSE-180
OACM5.1 C
-
-
-
-
-
0 24 48 72 96 120 1440
1
2
3
4
5
6
7
8
BHY EV
BHY SVIP
Time (days)
Cell
via
bil
ity (
a.u
.)
0 24 48 72 96 120 1440
1
2
3
4
5
6
7
8
Ca-SKI EV
Ca-SKI SVIP
Time (days)
Cell
via
bil
ity (
a.u
.)
*
0 24 48 72 96 120 1440
1
2
3
4
5
6
7
8
Colo680N EV
Colo680N SVIP
Time (days)
Cell
via
bil
ity (
a.u
.)
**
Time (hours)
Cell v
iab
ilit
y (
a.u
.)
0 24 48 72 96 120 1440
5
10
15
KYSE-180 EV
KYSE-180 SVIP
**
Supplemental Figure 2. Efficient restoration of SVIP in other cell lines. SVIP recovery upon transfection in BHY (head and neck cancer), Ca-Ski (cervical cancer), COLO-680N
(esophageal cancer) and KYSE-180 (esophageal cancer) cancer cell lines was validated by (A) qRT-PCR (biological replicates are shown, statistical analysis was performed using a two-
sided Student’s t test) and western blot (images are representative of at least three different assays). (B) Cell viability was determined by SRB assay (graphs are representative of three
independent experiments; statistical analysis was performed by comparing mean endpoints using two-tailed (C) In silico correlation of the expression of the cell cycle marker cyclin D1
depending on SVIP methylation and expression status in head and neck, esophageal, cervix and haematological cell lines. Samples whose SVIP expression was lower than the first quartile
were considered “SVIP low”, whereas samples with SVIP expression levels higher than the third quartile were considered “SVIP high”. Two-tailed U-Mann Whitney test was performed to
compare mRNA expression between the groups of cell lines and TCGA samples. (D) Comparison of the magnitude of SVIP overexpression in BB30-HNC cell line compared with two
unmethylated cell lines, Ca9-22 and Lb771 by qPCR.*P < 0.05, **P < 0.01, ***P<0.001.
Cell
via
bili
ty (
a.u
.)
Time (hours)
*
Cell
via
bili
ty (
a.u
.)
Time (hours)
Cell
via
bili
ty (
a.u
.)
Time (hours)
Cell
via
bili
ty (
a.u
.)
Time (hours)
A
B COLO-680N EV
COLO-680N SVIP
Ca-Ski EV
Ca-Ski SVIP BHY EV
BHY SVIP
KYSE-180 EV
KYSE-180 SVIP
SV
IP e
xpre
ssio
n (
a.u
.)
-ACTB
-SVIP
BHY E
V
BHY S
VIP
0.0
0.5
1.0
1.5
2.0
***
BHY EV
BHY SVIP
SV
IP e
xpre
ssio
n (
a.u
.)
-ACTB
-SVIP
Ca-
Ski
EV
Ca-
Ski
SVIP
0.0
0.5
1.0
1.5
2.0
***
Ca-Ski EV
Ca-Ski SVIP
SV
IP e
xpre
ssio
n (
a.u
.)
-ACTB
-SVIP
COLO
-680
N E
V
COLO
-680
N S
VIP
0.0
0.5
1.0
1.5
2.0
***
COLO-680N EV
COLO-680N SVIP
SV
IP e
xpre
ssio
n (
a.u
.)
-ACTB
-SVIP
KYSE-1
80 E
V
KYSE-1
80 S
VIP
0.0
0.5
1.0
1.5
2.0
***
KYSE-180 EV
KYSE-180 SVIP
0 24 48 72 96 120 1440
1
2
3
4
5
6
7
8
Ca-SKI EV
Ca-SKI SVIP
Time (days)
Cell
via
bil
ity (
a.u
.)
C D
-2
0
2
***
SVIP methylated (N=61)
SVIP unmethylated (N=138) CCND1
-3
-2
-1
0
1
2
3 ***
mR
NA
exp
ressio
n (
z-s
co
re)
mR
NA
expessio
n (
au)
SVIP low expression (N=50)
SVIP high expression (N=50)
CC
ND
1
mR
NA
expessio
n (
au)
Ca9
-22
Lb771
BB30
-HNC E
V
BB30
-HNC S
VIP
0
1
2
3
4
5
BB30-HNC EV
BB30-HNC SVIP
Ca9-22
Lb771
ns***
*** ****
*
mR
NA
exp
ressio
n (
a.u
.)
Ca9
-22
Lb771
BB30
-HNC E
V
BB30
-HNC S
VIP
0
1
2
3
4
5
BB30-HNC EV
BB30-HNC SVIP
Ca9-22
Lb771
ns***
*** ****
*
mR
NA
exp
ressio
n (
a.u
.)
mR
NA
expre
ssio
n (
a.u
.)
CC
ND
1
Supplemental Figure 3. Restoration of SVIP expression does not alter the expression of other proteins involved in ERAD machinery and also recapitulates the ER
stress situation in BHY and Ca-Ski cell lines. (A) Electron microscopy images of empty vector-transfected and SVIP-expressing BHY and Ca-Ski cell lines. All scale bars
represent 1µm. (B) Representative western blots of various proteins associated to UPR and protein folding in the ER after SVIP recovery in BHY and Ca-Ski cells. (C) In silico
correlation of the expression of three ER-related proteins (AMFR, DERL3 and XBP1) depending on SVIP methylation and expression status in head and neck, esophageal, cervix
and haematological cell lines and in head & neck TCGA cohort. Samples whose SVIP expression was lower than the first quartile were considered “SVIP low”, whereas samples
with SVIP expression levels higher than the third quartile were considered “SVIP high”. Two-tailed U-Mann Whitney test was performed to compare mRNA expression between the
groups of cell lines and TCGA samples. (D) Representative western blot of proteins involved in ERAD process after SVIP recovery in BB30-HNC, BHY and Ca-Ski cell lines. All western blots in B and D were performed in triplicate. (E) % of β-galactosidase-expressing cells in EV- and SVIP-transfected BB30-HNC cells. Statistical differences were determined using a two-sided Student’s t test.
A SVIP EV
BH
Y
Ca
-Ski
D
SVIP methylated (N=61)
SVIP unmethylated (N=138)
mR
NA
expessio
n (
au)
-2
0
2
4
(N=61
)
(N=13
8)
**
mR
NA
expessio
n (
au)
-2
0
2
4 ***
DE
RL
3
XB
P1
SVIP low expression (N=50)
SVIP high expression (N=50) DERL3
-4
-2
0
2
4
6** Low SVIP expression
High SVIP expression
mR
NA
expessio
n (
au)
XBP1
-4
-2
0
2
4 ***
mR
NA
expessio
n (
au)
DE
RL
3
XB
P1
-BiP
-PDI
-ERO1-Lα
-PERK
-IRE1α
-ACTB
-p-IRE1α
B C
BB30
-HNC E
V
BB30
-HNC S
VIP
0
10
20
30
40
50
ns
% O
f X
-Gal
po
sit
ive c
ell
s
Log[Thapsigargin (nM)]
% C
ell v
iab
ilit
y
0 1 2 3 4 5 60
20
40
60
80
100 BB30-HNC EV
BB30-HNC SVIP
**
E
-ACTB
-HRD1
-SEL1L
Supplemental Figure 4. ER-related drugs testing on BB30-HNC model. Determination of growth inhibitory effect by the SRB assay in empty
vector and SVIP- transfected BB30-HNC cells upon treatemnet with Bortezomib, 17-AAG, IPI-504, Eeyarestatin I and NMS-873. All assays were
performed in triplicate. Graphs are representative of the experiments. Statistical differences were determined by adjusting the curve to a third grade
polynomic equation followed by extra sum of squares F-test. ns, no significant.
Normalize of Row stats of Transform of Data 1
0.0 0.5 1.0 1.50
25
50
75
100 BB30-HNC EV
BB30-HNC SVIP
Log [Bortezomib (nM)]
Cell
via
bil
ity (
a.u
.)
ns
% C
ell
via
bili
ty
Log[Bortezomib (nM)]
-1 0 1 20
20
40
60
80
100BB30-HNC EV
BB30-HNC SVIP
Log [EerI (uM)]
% C
ell
via
bil
ity
ns
% C
ell
via
bili
ty
Log[Eeyarestatin I (µM)]
-2 -1 0 1 20
25
50
75
100 BB30-HNC EV
BB30-HNC SVIP
Log[17-AAG (uM)]
Cell
via
bil
ity (
a.u
.)
ns
% C
ell
via
bili
ty
Log[17-AAG (µM)]
-3 -2 -1 0 10
25
50
75
100
BB30-HNC SVIP
BB30-HNC EV
Log [IPI-504(uM)]
Cell
via
bil
ity (
a.u
.)
ns
% C
ell
via
bili
ty
Log[IPI-504 (µM)]
-5 -4 -3 -2 -10
25
50
75
100BB30 EV
BB30 SVIP
Log[NMS-873(uM)]
% C
ell
via
bil
ity
Log[Bay-876 (nM)]
% C
ell v
iab
ilit
y
0 1 2 3 4 50
20
40
60
80
100 BB30-HNC EV
BB30-HNC SVIP
ns
% C
ell
via
bili
ty
Log[NMS-873 (µM)]
Supplemental Figure 5. Metabolic consequences of SVIP recovery in BHY and Ca-Ski cancer cell lines. (A) Western blot of GLUT1 in empty vector and SVIP-restored
BHY and Ca-Ski cell lines. (B) SVIP mRNA expression significantly anticorrelates with GLUT1 mRNA expression in HNC, esophagus, cervix and haematological cell lines
(N=118). SVIP expression was considered low when the when it was lower or equal than the first quartile, while SVIP expression was considered high when it was equal or
higher than the third quartile. Expression was compared using a two-sided Mann-Whitney test. (C) In silico correlation of the expression of three metabolism-related proteins
(HK1 and UCP2) depending on SVIP methylation and expression status in head and neck, esophageal, cervix and haematological cell lines and in head & neck TCGA cohort.
Samples whose SVIP expression was lower than the first quartile were considered “SVIP low”, whereas samples with SVIP expression levels higher than the third quartile were
considered “SVIP high”. Two-tailed U-Mann Whitney test was performed to compare mRNA expression between the groups of cell lines and TCGA samples. (D) ATP production
in BHY and Ca-Ski cell lines upon SVIP transfection. Data were firstly normalized against protein amount, and then relativized against EV. Graph is representative of three
independent experiments. Values are represented as mean of six replicates. Statistical differences have been reported using two-sided Student’s t-test. (E) Mito Tracker median
fluorescence Intensity (MFI) was determined by flow cytometry. Mean SD of representative experiments is shown. (G). *P<0.05,**P<0.01,***P<0.001, ns no significant.
A B
-4
-2
0
2
4
High SVIP expression
Low SVIP expression
***
GL
UT
1 m
RN
A (
au
)
SVIP low expression (N=50)
SVIP high expression (N=50)
-GLUT1
-ACTB mR
NA
expre
ssio
n (
au)
0.00
0.25
0.50
0.75
1.00
1.25
1.50
**
ns
GLU
T1
expre
ssio
n (
a.u
.)
nm
ol A
TP
· µ
g p
rote
in-1
nm
ol A
TP
· µ
g p
rote
in-1
D E
BHY E
V
BHY S
VIP
0
25
50
75
100
125
150
175 *
Mit
otr
acker
(MF
I (a
u))
Mitotr
acker
MF
I (a
u)
Mitotr
acker
MF
I (a
u)
Transform of Ca-SKI Mitotracker
Ca-
Ski E
V
Ca-
Ski S
VIP
0
25
50
75
100
125
150
175
200
*
Mit
otr
acker
(MF
I (a
u))
0.0
0.5
1.0
1.5
2.0
2.5***
0.0
0.5
1.0
1.5
2.0
*
C
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)SVIP
low
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***m
RN
A e
xp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***m
RN
A e
xp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)M
ethyl
ated
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***m
RN
A e
xp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)SVIP
low
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
Cell lines TCGA samples
HK
1S
IRT
1U
CP
2
mR
NA
ex
pes
sio
n (au)
mR
NA
expessio
n (au)
mR
NA
ex
pes
sio
n (au)
mR
NA
expessio
n (au)
mR
NA
expessio
n (au)
mR
NA
expessio
n (au)
mR
NA
exp
essio
n (a
u)
mR
NA
exp
essio
n (a
u)
mR
NA
ex
pe
ss
ion
(a
u)
mR
NA
exp
essio
n (a
u)
mR
NA
exp
essio
n (a
u)
mR
NA
exp
essio
n (a
u)
SVIP methylated (N=61)
SVIP unmethylated (N=138)
SVIP low expression (N=50)
SVIP high expression (N=50)
SVIP methylated (N=190)
SVIP unmethylated (N=274)
SVIP low expression (N=116)
SVIP high expression (N=116)
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)SVIP
low
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***m
RN
A e
xp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP lo
w
SVIP h
igh
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP lo
w
SVIP h
igh
-4
-2
0
2
4 ***
Cell lines TCGA samples
HK
1S
IRT
1U
CP
2
mR
NA
ex
pe
ss
ion
(a
u)
mR
NA
exp
essio
n (au
)m
RN
A e
xp
es
sio
n (
au
)
mR
NA
exp
essio
n (au
)m
RN
A e
xp
essio
n (au
)m
RN
A e
xp
essio
n (au
)
mR
NA
expessio
n (au)
mR
NA
expessio
n (au)
mR
NA
ex
pe
ss
ion
(a
u)
mR
NA
expessio
n (au)
mR
NA
expessio
n (au)
mR
NA
expessio
n (au)
SVIP methylated (N=61)
SVIP unmethylated (N=138)
SVIP low expression (N=50)
SVIP high expression (N=50)
SVIP methylated (N=190)
SVIP unmethylated (N=274)
SVIP low expression (N=116)
SVIP high expression (N=116)
HK
1
UC
P2
SVIP methylated (N=61)
SVIP unmethylated (N=138)
SVIP low expression (N=50)
SVIP high expression (N=50)
SVIP methylated (N=190)
SVIP unmethylated (N=274)
SVIP low expression (N=116)
SVIP high expression (N=116)
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***m
RN
A e
xp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)M
ethyl
ated
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***m
RN
A e
xp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)M
ethyl
ated
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***m
RN
A e
xp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
Cell lines TCGA samples
HK
1S
IRT
1U
CP
2
mR
NA
ex
pes
sio
n (au)
mR
NA
expessio
n (au)
mR
NA
ex
pes
sio
n (au)
mR
NA
expessio
n (au)
mR
NA
expessio
n (au)
mR
NA
expessio
n (au)
mR
NA
exp
essio
n (a
u)
mR
NA
exp
essio
n (a
u)
mR
NA
ex
pe
ss
ion
(a
u)
mR
NA
exp
essio
n (a
u)
mR
NA
exp
essio
n (a
u)
mR
NA
exp
essio
n (a
u)
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***m
RN
A e
xp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***m
RN
A e
xp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 **
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4ns
SVIP
low
SVIP
hig
h-4
-2
0
2
4ns
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
SVIP
low
SVIP
hig
h
-2
0
2
4 ***
mR
NA
exp
ressio
n (
a.u
.)
Met
hylat
ed
Unm
ethyl
ated
-4
-2
0
2
4 ***
SVIP
low
SVIP
hig
h-4
-2
0
2
4 ***
Cell lines TCGA samples
HK
1S
IRT
1U
CP
2
mR
NA
ex
pes
sio
n (au)
mR
NA
expessio
n (au)
mR
NA
ex
pes
sio
n (au)
mR
NA
expessio
n (au)
mR
NA
expessio
n (au)
mR
NA
expessio
n (au)
mR
NA
exp
essio
n (a
u)
mR
NA
exp
essio
n (a
u)
mR
NA
ex
pe
ss
ion
(a
u)
mR
NA
exp
essio
n (a
u)
mR
NA
exp
essio
n (a
u)
mR
NA
exp
essio
n (a
u)
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0*
mR
NA
exp
ressio
n (
a.u
.)
CCND1
mR
NA
expessio
n (
au)
SVIP methylated (N=61)
SVIP unmethylated (N=138)
SVIP low expression (N=50)
SVIP high expression (N=50)
SVIP methylated (N=190)
SVIP unmethylated (N=274)
SVIP low expression (N=116)
SVIP high expression (N=116)
0 1 2 3 4 50.0
0.2
0.4
0.6
0.8
1.0
**
Log Rank p-value=0.0046
HR(95% CI) = 2.006 (1.239-3.247)
Dis
ease fre
e s
urv
ival SVIP high expression (N=99)
SVIP low expression (N=99)
Years since diagnosis
0 1 2 3 4 50.0
0.2
0.4
0.6
0.8
1.0
**
Log Rank p-value=0.0077
HR(95% CI) = 1.678 (1.147-2.455)
Years since diagnosis
Overa
ll surv
ival
SVIP high expression (N=131)
SVIP low expression (N=131)
Overa
ll surv
ival
0 24 48 72 96 120
144
168
192
216
0.0
0.2
0.4
0.6
0.8
1.0
ns
GLUT1 high
GLUT1 low
Time (months)
Fra
cti
on
su
rviv
al
Log Rank p-value=0.054
HR(95% CI) = 1.461(0.994-2.146)
Time (months)
GLUT1 high expression (N=131)
GLUT1 low expression (N=131)
0 12 24 36 48 60 72
0.0
0.2
0.4
0.6
0.8
1.0
ns
GLUT1 high
GLUT1 low
Time (months)
Fra
cti
on
su
rviv
al
Dis
ease
fre
e s
urv
ival
Time (months)
Log Rank p-value=0.8193
HR(95% CI) = 1.058(0.651-1722)
GLUT1 low expression (N=99)
GLUT1 high expression (N=99)
Supplemental figure 6. SVIP epigenetically-deficient head & neck TCGA tumors recapitulate the phenotype observed in SVIP-recovered cell lines. (A) In silico correlation of
the expression of XBP1 and DERL3 depending on SVIP expression status in head & neck TCGA cohort. (B) GLUT1 expression analysis in head and neck, esophageal and cervical
TCGA samples. (B) GLUT1 expression analysis in head and neck, esophageal and cervical TCGA samples depending on SVIP expression levels. (C) In silico correlation of the
expression of HK1 and UCP2 depending on SVIP expression status in head & neck TCGA cohort. (D) Kaplan Meier analysis of disease free survival (left) and overall survival (right)
considering SVIP expression in head and neck primary tumors of the TCGA data set. P- value corresponds to log rank test. Results of univariate Cox regression analysis are shown
by hazard ratio (HR) and 95% confidence interval (CI). (E) Kaplan Meier analysis of disease free survival (left) and overall survival (right) considering GLUT1 expression in head and
neck primary tumors of the TCGA data set. P- value corresponds to log rank test. Results of univariate Cox regression analysis are shown by hazard ratio (HR) and 95% confidence
interval (CI). (F) In silico correlation of the expression of CCND1 depending on SVIP expression status in head & neck TCGA cohort. Samples in all panels were divided in quartiles
according to SVIP or GLUT1 expression. Samples whose SVIP/GLUT1 expression was lower than the first quartile were considered “low”, whereas samples with expression levels
higher than the third quartile were considered “high. Two-tailed U-Mann Whitney test was performed to compare gene expression between the studied groups of cell lines and TCGA
samples. *P<0.05,**P<0.01,***P<0.001, ns no significant.
C SVIP methylated (N=61)
SVIP unmethylated (N=138)
-2
0
2
4**
mR
NA
exp
ress
ion
(a.
u.)
mR
NA
ex
pe
ss
ion
(a
u)
mR
NA
expessio
n (au)
-2
0
2
4
(N=61
)
(N=13
8)
**
mR
NA
expessio
n (au)
-2
0
2
4 ***
AM
FR
DE
RL
3X
BP
1
SVIP low expression (N=50)
SVIP high expression (N=50)AMFR
-2
-1
0
1
2
3
4 *
mR
NA
expessio
n (au)
DERL3
-4
-2
0
2
4
6** Low SVIP expression
High SVIP expression
mR
NA
ex
pe
ss
ion
(a
u)
XBP1
-4
-2
0
2
4 ***
mR
NA
expessio
n (au)
AM
FR
DE
RL
3X
BP
1
SVIP methylated (N=190)
SVIP unmethylated (N=274)
2.5
3.0
3.5
4.0**
mR
NA
expessio
n (au)
0
1
2
3
4 ***
mR
NA
ex
pe
ss
ion
(a
u)
2.5
3.0
3.5
4.0
4.5 ***
mR
NA
expessio
n (au)
AM
FR
DE
RL
3X
BP
1
SVIP low expression (N=116)
SVIP high expression (N=116)
2.5
3.0
3.5
4.0***
mR
NA
ex
pe
ss
ion
(a
u)
0
1
2
3
4 ***
mR
NA
exp
ressio
n (
a.u
.)m
RN
A e
xp
es
sio
n (
au
)
XBP1
2.5
3.0
3.5
4.0
4.5 ***
mR
NA
exp
ressio
n (
a.u
.)m
RN
A e
xp
es
sio
n (
au
)
AM
FR
DE
RL
3X
BP
1
Cell lines TCGA samples
C SVIP methylated (N=61)
SVIP unmethylated (N=138)
-2
0
2
4**
mR
NA
exp
ress
ion
(a.
u.)
mR
NA
ex
pe
ss
ion
(a
u)
mR
NA
expessio
n (au)
-2
0
2
4
(N=61
)
(N=13
8)
**
mR
NA
expessio
n (au)
-2
0
2
4 ***
AM
FR
DE
RL
3X
BP
1
SVIP low expression (N=50)
SVIP high expression (N=50)AMFR
-2
-1
0
1
2
3
4 *m
RN
A e
xpessio
n (au)
DERL3
-4
-2
0
2
4
6** Low SVIP expression
High SVIP expression
mR
NA
ex
pe
ss
ion
(a
u)
XBP1
-4
-2
0
2
4 ***
mR
NA
expessio
n (au)
AM
FR
DE
RL
3X
BP
1
SVIP methylated (N=190)
SVIP unmethylated (N=274)
2.5
3.0
3.5
4.0**
mR
NA
expessio
n (au)
0
1
2
3
4 ***
mR
NA
ex
pe
ss
ion
(a
u)
2.5
3.0
3.5
4.0
4.5 ***
mR
NA
expessio
n (au)
AM
FR
DE
RL
3X
BP
1
SVIP low expression (N=116)
SVIP high expression (N=116)
2.5
3.0
3.5
4.0***
mR
NA
ex
pe
ss
ion
(a
u)
0
1
2
3
4 ***
mR
NA
exp
ressio
n (
a.u
.)m
RN
A e
xp
es
sio
n (
au
)
XBP1
2.5
3.0
3.5
4.0
4.5 ***
mR
NA
exp
ressio
n (
a.u
.)m
RN
A e
xp
es
sio
n (
au
)
AM
FR
DE
RL
3X
BP
1
Cell lines TCGA samples
C SVIP methylated (N=61)
SVIP unmethylated (N=138)
-2
0
2
4**
mR
NA
exp
ress
ion
(a.
u.)
mR
NA
ex
pe
ss
ion
(a
u)
mR
NA
expessio
n (au)
-2
0
2
4
(N=61
)
(N=13
8)
**
mR
NA
expessio
n (au)
-2
0
2
4 ***
AM
FR
DE
RL
3X
BP
1
SVIP low expression (N=50)
SVIP high expression (N=50)AMFR
-2
-1
0
1
2
3
4 *
mR
NA
expessio
n (au)
DERL3
-4
-2
0
2
4
6** Low SVIP expression
High SVIP expression
mR
NA
ex
pe
ss
ion
(a
u)
XBP1
-4
-2
0
2
4 ***
mR
NA
expessio
n (au)
AM
FR
DE
RL
3X
BP
1
SVIP methylated (N=190)
SVIP unmethylated (N=274)
2.5
3.0
3.5
4.0**
mR
NA
expessio
n (au)
0
1
2
3
4 ***
mR
NA
ex
pe
ss
ion
(a
u)
2.5
3.0
3.5
4.0
4.5 ***
mR
NA
expessio
n (au)
AM
FR
DE
RL
3X
BP
1
SVIP low expression (N=116)
SVIP high expression (N=116)
2.5
3.0
3.5
4.0***
mR
NA
ex
pe
ss
ion
(a
u)
0
1
2
3
4 ***m
RN
A e
xp
ressio
n (
a.u
.)m
RN
A e
xp
es
sio
n (
au
)
XBP1
2.5
3.0
3.5
4.0
4.5 ***
mR
NA
exp
ressio
n (
a.u
.)m
RN
A e
xp
es
sio
n (
au
)
AM
FR
DE
RL
3X
BP
1
Cell lines TCGA samples
SL
C2A
1 e
xp
ressio
n (
a.u
.)
HNC S
VIP
exp
low
HNC S
VIP
exp
hig
h
Eso
phagus
SVIP
exp
low
Eso
phagus
SVIP
exp
hig
h
Cer
vix
SVIP
exp
low
Cer
vix
SVIP
exp
hig
h
-2
0
2
4
6
8*** *** ***
GLU
T1
mR
NA
expre
ssio
n (
a.u
.)
SVIP low expression SVIP high expression
A B C
D E F
UCP2 HK1
CCND1
DERL3 XBP1
Supplementary Table 1: List of differentially expressed proteins represented for at least two
peptides in SILAC analysis upon SVIP transfection in BB30-HNC cells.
A. Upregulated upon SVIP overexpression
ID Gene Fold change p-value
FXR1 32.1428571 0.0299974
DHCR7 15.1020408 1.6286E-05
RTCA 12.8333333 0.04864284
VIM 10.617284 0.04760836
CNN3 10.1557632 0.02364341
HMGCS1 8.80090498 0.003165
LPCAT1 7.62942779 0.04977868
COPS8 7.34375 0.04210733
AP3S1 7.26744186 0.0199053
ABCD3 6.91333982 0.03893685
SLC25A24 6.82983683 0.04516253
CAT 5.85714286 0.00127963
PAIP1 5.55555556 0.0228933
VAT1 5.52857143 0.00319767
DNM2 5.37634409 0.04575119
IDH1 5.20661157 0.03582669
ITGA2 5.10416667 0.04200355
TARS 5.03846154 0.04664995
CTTN 4.95495495 0.02048994
ACAT2 4.60063898 0.00282376
RBM12 4.57142857 0.00441503
EIF2AK2 4.50980392 0.04177745
ERAP1 4.48514851 0.04793058
DCDC5 4.47368421 0.01507317
OSBP 4.44444444 0.01913367
S100A13 4.41176471 0.04020444
PPP2R2A 4.375 0.00362953
TKT 4.21232877 0.01032101
DDAH1 4.20588235 0.02980616
CYB5A 4.12612613 0.02691284
CUL4B 4.07619048 0.03584337
GDI1 3.93364929 0.02618891
PRKCDBP 3.92215569 0.01801815
PGM1 3.84615385 0.02005603
BASP1 3.70588235 0.00228101
SUB1 3.66666667 0.01454491
RRM2 3.6 0.00576178
G6PD 3.47692308 0.01762289
DTD1 3.4502924 0.03773559
MCTS1 3.4351145 0.0028799
ID Gene Fold change p-value
OGFR 3.42857143 0.04988295
PDIA6 3.41463415 0.01552488
KRT8 3.36585366 0.03671742
SUCLG2 3.34650856 0.01987182
GNAS 3.34117647 0.01566528
AKR1C1 3.32653061 0.04096684
RAB10 3.27058824 0.04417397
MTHFD1 3.25203252 0.01294349
RNPS1 3.24817518 0.03883771
ADIRF 3.22988506 0.01315107
GNB2 3.2183908 0.00195006
APEX1 3.2173913 0.01636789
NDUFB11 3.20512821 0.01195103
DENR 3.18181818 0.006425
PDPN 3.16363636 0.03425198
HADHB 3.08457711 0.04654997
UGDH 3.03478261 0.01298571
NUDT5 3.02325581 0.00117976
PSMF1 2.97752809 0.00378019
SLC25A5 2.96482412 0.01368587
MVP 2.92517007 0.03508987
GNAI2 2.92307692 0.00454258
APP 2.91727141 0.01900122
CORO1C 2.87096774 0.01134609
AKR1B1 2.85714286 0.03850582
LTA4H 2.84415584 0.03016842
PAFAH1B3 2.77966102 0.0310627
ACSL3 2.76960784 0.02925421
VCL 2.75630252 0.04905471
S100A6 2.7480916 0.00856122
SLC25A22 2.73737374 0.0198122
PGK1 2.72857143 0.04693298
CD82 2.72821577 0.0367791
MARS 2.7027027 0.0370125
HIBADH 2.68421053 0.00494985
MT-CO2 2.67409471 0.04651879
DDX1 2.62626263 0.03135003
ERP29 2.62365591 0.03317915
MAPRE1 2.59722222 0.00459747
APEH 2.5748503 0.00782955
AIFM1 2.57425743 0.02232868
KIF5B 2.5462963 0.02511471
NUMA1 2.5 0.02771502
PFKFB2 2.48863636 0.0479245
ECI1 2.47191011 0.01217441
ID Gene Fold change p-value
PREP 2.46808511 4.0259E-05
RALY 2.46153846 0.03930777
MBOAT7 2.45901639 0.039528
YBX3 2.42105263 0.02324571
PRDX5 2.4137931 0.00031503
GSTO1 2.36538462 0.04432432
CLNS1A 2.35443038 0.04334824
HADHA 2.32365145 0.00628803
SRI 2.31944444 0.01029654
ARHGAP1 2.31428571 0.00012989
TM9SF4 2.29885057 0.04998547
AARS 2.29032258 0.0073872
APMAP 2.28571429 0.00265961
RAB6C 2.25663717 0.04348661
ARPC2 2.24489796 0.03437383
RBBP4 2.17898833 0.04339225
TSPO 2.17333333 0.03546801
CLIC4 2.1541502 0.0188887
SARS 2.15 0.03363727
PRDX6 2.12765957 0.03805794
NUDT21 2.10918114 0.04786461
PKP1 2.09146341 0.03078166
RAVER1 2.06293706 0.04912038
CDC37 2.05555556 0.03594591
EIF1 2.05417607 0.02626985
B. Downregulated upon SVIP overexpression
ID Gene Fold change p-value
SIX5 0.02891892 0.02429929
TYMP 0.03857143 0.01735944
GSTP1 0.0401626 0.04078133
HSPB1 0.04258065 0.01581999
HLA-C 0.05031447 0.04112482
HLA-B 0.26961326 0.02763397
BICD2 0.27108434 0.00847096
ETFA 0.34016393 0.04288903
ETFB 0.35632184 0.00542287
APRT 0.36363636 0.04489244
ARPC5L 0.37623762 0.04469599
CLTB 0.38888889 0.04566731
PC 0.41958763 0.04420214
SLC2A1 0.46545455 0.0214964
Supplementary Table 2: Association between clinicopathological characteristics and SVIP
methylation status in the TCGA head & neck squamous cell carcinoma cohort.
CHARACTERISTICS SVIP methylation status
Unmethylated Methylated P*
N (%) N (%) N (%)
Age (years)
< 50 48 (17%) 32 (67%) 16 (33%) 0.264
> 50 234 (83%) 135 (58%) 99 (42%)
Gender
Male 200 (71%) 130 (65%) 70 (35%) 0.003**
Female 82 (29%) 37 (45%) 45 (55%)
Disease stage at diagnosis
I 11 (4%) 7 (64%) 4 (36%) 0.207
II 55 (19%) 27 (49%) 28 (51%)
III 67 (24%) 37 (55%) 30 (45%)
IV 136 (48%) 88 (65%) 48 (35%) Unknown 13 (5%) 8 (62%) 5 (38%)
Tumor site
Oral cavity 104 (37%) 41 (39%) 63 (61%) <0.001***
Tongue 92 (33%) 55 (60%) 37 (40%)
Tonsil 16 (6%) 15 (94%) 1 (6%) Pharynx 10 (4%) 9 (90%) 1 (10%)
Larynx 59 (21%) 47 (80%) 12 (20%) Unknown 1 (1%) 0 (0%) 1 (100%)
Smoking habit
Current / Former smoker 15 (5%) 7 (47%) 8 (53%) 0.287 Never smoker 185 (66%) 109 (59%) 76 (41%)
Unknown 82 (29%) 51 (62%) 31 (38%)
Drinking habits
Yes 177 (63%) 114 (64%) 63 (36%) 0.031* No 100 (35%) 51 (51%) 49 (49%)
Unknown 5 (2%) 2 (40%) 3 (60%)
HPV status
Negative 49 (17%) 37 (75%) 12 (25%) 0.358 Positive 23 (8%) 20 (87%) 3 (13%)
Unknown 210 (75%) 110 (52%) 100 (48%)
Treatment strategy
Surgery 94 (34%) 55 (58%) 39 (42%) 0.096
Chemotherapy 40 (14%) 31 (12%) 9 (23%)
Radiotherapy 32 (11%) 22 (69%) 10 (31%)
Unknown 116 (41%) 59 (51%) 57 (49%) Treatment outcome
Complete remission/Response 171 (61%) 108 (63%) 63 (37%) 0.650
Stable disease 7 (2%) 5 (71%) 2 (29%)
Progressive disease 44 (16%) 25 (57%) 19 (43%)
Unknown 60 (21%) 29 (48%) 31 (52%) Survival status
Alive 200 (71%) 127 (63%) 73 (37%) 0.024*
Dead 82 (29%) 40 (49%) 42 (51%)
*P-value represents Fisher's exact test or Chi-square function whenever required: statistical significance under 0.05*, 0.01** and 0.001***
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
SVIP SIGMA HPA039807
phospho-IRE1α (Ser724) Thermofisher PA1-16927
p21 CST 2947
eIF2α CST 5324
phospho-eIF2α (Ser51) CST 3398
SEL1L Abcam Ab78298
SYVN1 (Hrd1) CST 14773
IRE1α CST 14C10
PERK CST D11A8
ERO1-Lα CST #3264
PDI CST C81H6
BECLIN1 CST D40C5
p62/SQTM CST #5114
LC3BI/II CST D3U4C
PARP BD Pharmingen 556362
Caspase 3 SCBT H-277
HM13 Abcam ab190253
REEP5 SIGMA HPA003895
CD82 Abcam ab66400
NSDHL Abcam ab190353
SLC25A5 CST #14671
SUCLG2 Abcam ab96172
NDUFB8 (CI) Abcam ab110411
MTCO2 (CIV) Abcam ab110411
SLC2A1 CST D3J3A
αTubulin-HRP Abcam 40742
LMNB1 Abcam ab16048
ACTB -HRP SIGMA A3854
Anti-rabbit HRP SIGMA A0545
Anti-mouse HRP GE HEALTHCARE NA9310
Bacterial and Virus Strains
E. coli DH5α Thermofisher 18265017
Lentivirus produced with PsPPAX and PDM2 plasmids - -
Chemicals, Peptides, and Recombinant Proteins
5’-azacytidine SIGMA A2385
Sulforhodamine B SIGMA S1402
Polyethylene glycol 300 SIGMA 90878-1L
NMP SIGMA 494496-1L
GoScript Promega A5001
Sybr green Thermofisher 4312704
jetPrime polyplus Polyplus Ref114
Bortezomib LC laboratories B-1408
Eeyarestatin I SIGMA E1286
17-AAG SIGMA A8476
IPI-504 Medchem HY-10210
Thapsigargin SIGMA T9033
SILAC-Lys8-Arg10-Kit media SILANTES 282986444
Bay-876 Sigma SML1774
NMS-873 MedChemExpress HY-15713
Oligomycin SIGMA 75351
FCCP SIGMA C2920
Rotenone A SIGMA R8875
Antimycin A SIGMA A8674
Hydrazine Sigma 53847
NAD+ Roche - Sigma 10127990001
LDH Roche - Sigma 10107085001
dATP NEBiolabs N0440S
Mito Tracker Thermofisher M22426
MitoSox Thermofisher M36008
U-13
C6 labeled glucose Sigma 389374
Critical Commercial Assays
EZ-DNA methylation Gold Kit Zimo research D5006
Nucleospin Gel and PCR Clean-up Machery Nagel 740609.250
Nucleospin 96 plasmid Machery Nagel 740625.24
Maxwell RSC simply RNA tissue Promega AS1340
Seahorse XF24 plates Agilent 100777-004
DC protein assay BioRad 5000116
Cell titer Glo Promega G7571
Glucose HK CP ABX diagnostics A11A01667
Experimental Models: Cell Lines
BB30-HNC Sanger CVCL_1076
BHY DSMZ CVCL_1086
Ca9-22 Sanger CVCL_1102
LB-771 Sanger CVCL_1369
Ca-Ski ATCC CVCL_1100
HeLa ATCC CVCL_0030
SW756 ATCC CVCL_1727
COLO-680N DSMZ CVCL_1131
KYSE-180 DSMZ CVCL_1349 OACM 5.1 C SIGMA 11012006 HEK-293T ATCC CVCL_0063
Experimental Models: Organisms/Strains
NU/NU Nude Mice INVIGO
Oligonucleotides
BS SVIP F TTTTTTGGAATTTAGGTTTTGG This paper
BS SVIP R AAAAACACAACCCCATAAAAC This paper
Cloning SVIP S AAAAAAAAGAATTCGCCGCCACCATGGGGCTGTGTTTTCCTTGTCCCGGGGAGTCCGCGCCTC
This paper
Cloning SVIP AS AAAAAAAAGGATCCTTACTTATCGTCGTCATCCTTGTAATCGCCGGAGCCTGAAACTGTCCACCTAAGTCCACCTTCTGGTGGGGGCCCGGATGTAGCAA
This paper
sh1_SVIP S gatccGACTTAGGTGGACAGTTTCTTCAAGAGAGAAACTGTCCACCTAAGTCTTTTTTACGCGTg
This paper
sh1_SVIP AS aattcACGCGTAAAAAAGACTTAGGTGGACAGTTTCTCTCTTGAAGAAACTGTCCACCTAAGTCg
This paper
sh4_SVIP S gatccGGCTAGACTTTTCATTAAATTCAAGAGATTTAATGAAAAGTCTAGCCTTTTTTACGCGTg
This paper
sh4_SVIP AS aattcACGCGTAAAAAAGGCTAGACTTTTCATTAAATCTCTTGAATTTAATGAAAAGTCTAGCCg
This paper
Scr_SVIP S gatccGCGCAGAACAAATTCGTCCATTCAAGAGATGGACGAATTTGTTCTGCGTTTTTTACGCGTg
PMID: 28881673
Scr_SVIP AS aattcACGCGTAAAAAACGCAGAACAAATTCGTCCATCTCTTGAATGGACGAATTTGTTCTGCGCg
PMID: 28881673
q SVIP F AAAGAGCAAAGCTTGCAGAG This paper
q SVIP R GAAACTGTCCACCTAAGTCCAC This paper
q GAPDH F TGCACCACCAACTGCTTAGC This paper
q GAPDH R GGCATGGACTGTGGTCATGAG This paper
qSLC25A5 F CTACAAATCTGATGGATTAAGGG This paper
qSLC25A5 R ATGATGTCAGTTCCTTTGCG This paper
qSUCLG2 F CTGATCCTAAGGTTGAAGCCA This paper
qSUCLG2 R CGCTGTTGTTGAGTATCTTCTG This paper
qNDUFB8 F CCCTATCCTAGGACCCCAGA This paper
qNDUFB8 R TCCACACGGTTCCTGTTGTA This paper
qMT-CO2 F TTCATGATCACGCCCTCATA This paper
qMT-CO2 R TAAAGGATGCGTAGGGATGG This paper
qHM13 F CAGACATGCCTGAAACAATCA This paper
qHM13 R GATTCCCAGCACGAAGAAAT This paper
qREEP5 F CTTCATCGCTCTTGGTGTCA This paper
qREEP5 R GGTCAGCCACTGGGTATCAT This paper
qCD82 F TGAGGACTGGCCTGTGTACC This paper
qCD82 R TTGGGACCTTGCTGTAGTC This paper
qNSDHL F GACCTTCCCTATGCCATGAA This paper
qNSDHL R CCAAGTTCTTCCCATTTCCA This paper
qSLC2A1 F CATCAACGCTGTCTTCTATTACTC This paper
qSLC2A1 R ATGCTCAGATAGGACATCCA This paper
Recombinant DNA
PsPax2 Addgene 12260
PDM2.G Addgene 12259
pLVX-Ires-ZSgreen1 Clonetech 632187
pLVX-shRNA2 Clonetech 632179
Software and Algorithms
cgds-R package The R project
Bioedit Bio-Soft
SPSS IBM
GraphPad Prism 5 GraphPad Software, Inc
Methyl Primer Express v1.0 Applied Biosystems
Primer3 Source Forge
UCSC genome browser UCSC
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