Instructions for use
Title Eva1 Maintains the Stem-like Character of Glioblastoma-Initiating Cells by Activating the Noncanonical NF-κBSignaling Pathway
Author(s) Ohtsu, Naoki; Nakatani, Yuka; Yamashita, Daisuke; Ohue, Shiro; Ohnishi, Takanori; Kondo, Toru
Citation Cancer research, 76(1), 171-181https://doi.org/10.1158/0008-5472.CAN-15-0884
Issue Date 2016-01
Doc URL http://hdl.handle.net/2115/63977
Type article (author version)
Additional Information There are other files related to this item in HUSCAP. Check the above URL.
File Information Ohtsu_et_al-revised-Supplementary_information.pdf (Supplementary Information)
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
1
Supplementary Information
Supplementary Materials and Methods
Antibody
Rabbit anti-Eva1 polyclonal antibody was generated by the custom antibody service of
the Medical & Biological Laboratories Co. Ltd (MBL) (Nagoya, Japan). In brief, the
synthetic peptide, CPMSGRFKDRVSWDGNPE (AA 86−102, corresponding to human
Eva1), was conjugated with KLH (Keyhole limpet hemocyanin), mixed with the
Freund’s complete adjuvant and injected into the intradermal of rabbits (Female New
Zealand White). Two weeks later, the KLH-conjugated peptide with the Freund’s
incomplete adjuvant was injected 7 times weekly. The antibody was purified from the
serum using the synthetic peptide-conjugaed affinity column.
ELISA (Enzyme-linked immunosorbent assay)
The microtiter plates were coated with the non-conjugated peptide (1μg/ml) in PBS at
4 ℃ overnight. After blocking with the Blocking buffer (MBL), the
antibody-conjugated plates were incubated with the purified antibody with indicated
2
concentration, following with the HRP conjugated goat anti-rabbit IgG (MBL). The
plates were then reacted with the substrate solution (MBL) and the absorbance was
measured using a Microplate reader Sunrise (TECAN).
Vector construction
Mouse eva1 cDNA was inserted into the pcDNA3-2xFLAG-c vector (Invitrogen),
resulting in pcDNA3-mEva1-2xFLAG-c. The following oligonucleotide DNA primers
were synthesized to amplify mouse full eva1 cDNAs: the 5' primer was
5'-AGAATTCGCCACCATGTATGGCAAGAGCCCCGC-3', and the 3' primer was
5'-ACTCGAGGTCTGTATCTTCCACAAAAACA-3'.
Mouse relb cDNA was cloned as described previously (7) and was inserted into
the pcDNA3-2xFLAG-c vector (Invitrogen), resulting in pcDNA3-RelB-2xFLAG-c.
The following oligonucleotide DNA primers were synthesized to amplify full relb
cDNA: the 5' primer was
5'-GGGAATTCACCGCCATGCCGAGTCGCCGCGCTGC-3', and the 3' primer was
5'-GGCTCGAGCGTGGCTTCAGGCCCTGGAG-3'.
3
RT-PCR
RT-PCR was performed as described previously (14). The following oligonucleotide DNA
primers were synthesized: for aldh1a3, as follows: 5’ primer,
5’-AAGAAGGAAGGGGCCAAGCT-3’; 3’ primer,
5’-TGGTGACAGTTTTCACTTCTGT-3’; for hey1, the 5’ primer was
5’-GCGGACGAGAATGGAAACTTG-3’, and the 3’ primer was
5’-AGTCCTTCAATGATGCTCAGAT-3’; for prdm16, the 5’ primer was
5’-GAGAAGCTGGAGAGCTTTGCA -3’, and the 3’ primer was
5’-AGGTTGGTCTGCTGCCCGAA-3’; for notch4, the 5’ primer was
5’-TGTGGCTGCCCCCTGGTTTCA-3’, and the 3’ primer was
5’-GTGTCACCCCATCAGGTCCAC-3’; for abcg2, the 5’ primer was
5’-AACTTACAGTTCTCAGCAGCTCTT-3’, and the 3’ primer was
5’-GCTGATGAATGGAGAAGATGATTGTT-3’. The primers for gapdh were as described
previously (42).
4
Quantitative RT-PCR (qRT-PCR)
qRT-PCR analysis was performed using Fast SYBR Green Master Mix (Applied
Biosystems) with a StepOne Real-Time PCR Systems (Applied Biosystems). The PCR
condition was as follows: 10 minutes at 95°C, followed by 40 cycles of 95°C for 15 sec
and 60°C for 1 min. All of the expression values were normalized against β-actin
mRNA expression levels. Relative gene expression was calculated using the 2-ΔΔCt
method (10). The following oligonucleotide DNA primers were synthesized: for mouse
eva1; 5’ primer, 5’-TGGGGCCACGACTGGCTTTCC-3’; 3’ primer, 5’-
ACAGGGGCAAAGCTGGAGAAAGTG-3’; for human eva1: 5’ primer,
5’-TGGGGCCAAGGCTGGGTTTCC-3’; 3’ primer, 5’-
TGAGCAGTTTGTATTCTACTACCA-3’; for human cd15: 5’ primer,
5’-ACTCGCAGCACCTGGATTAT-3’; 3’ primer,
5’-CGAGGAAAAGCAGGTACGAG-3’; for human cd49f: 5’ primer,
5’-TCATGGATCTGCAAATGGAA-3’; 3’ primer,
5’-AGGGAACCAACAGCAACATC-3’; for human sox2: 5’ primer
5’-CCATGCAGGTTGACACCGTTG-3’; 3’ primer,
5
5’-TCGGCAGACTGATTCAAATAATACAG-3’; for β-actin: 5’primer,
5’-AGAAGAGCTATGAGCTGCCTGACG-3’; 3’ primer,
5’-TACTTGCGCTCAGGAGGAGCAATG-3’.
Limiting dilution assay
hGICs that were transfected with control, eva1-, eva1sh- or relbsh-expression vectors
were single cell-sorted into each well of 96-well plate using the FACS Aria II (BD).
After confirming the sorting, cells were cultured for 10 days and the colony-forming
wells were counted.
6
Supplementary Figure Legends
Figure S1. Eva1 expression level in mouse GICs, human GIC and glioma tissues
A, Relative eva1 expression in mouse GICs, NSCL61 and OPCL61, compared with
their parental control cells, NSCs and OPCs. B, Relative eva1 expression in human
GICs (hGICs), E2, E3 and E6, compared with human NSC. C, Relative eva1 expression
in GBM, anaplastic oligodendroglioma (AO), anaplastic oligoastrocytoma (AOA),
oligodendroglioma (OLI) and control brain (CB), normalized to β-actin. Error bar
indicates ±SD. t test was used for statistical significance. * p<0.05 and ** p<0.01
significantly different from control cells.
Figure S2. Generation of rabbit polyclonal anti-Eva1 antibodies
A, Alignment of human and mouse Eva1 amino-acid sequences. Bold characters,
arrowheads and box indicate the signal sequence, Cystein residues and transmembrane
domain, respectively. Underline shows the peptide antigen sequence. B, ELISA assay of
the anti-Eva1 antibody to the peptide antigen. C, mNSC, NSCL61 and OPCL61 were
7
immunostained for Eva1 (green). Nuclei were counterstained with DAPI (blue). Scale
bar: 50 μm. D, Sagittal sections from the brains of E18, P1, and P100 mice were
immunolabeled for Eva1 (Green) and Nestin (Red). Nuclei were counterstained with
DAPI (Blue). V: ventricle. Scale bar: 100 m.
Figure S3. Knockdown efficiency of eva1sh-expression vectors
FLAG-tagged mouse (A) and human (B) Eva1 expression vectors were transfected with
a contsh or eva1sh expression vector into Cos7 cells. Cell extracts were harvested 2 d
after transfection, and were analyzed by Western blotting using anti-Eva1, anti-FLAG
and anti-GAPDH (loading control) antibodies.
Figure S4. Self-renewal activity of eva1- and eva1sh-expressing hGICs
A, Overexpression of eva1 increased self-renewal activity of E3 and E6. B, Eva1
knockdown decreased self-renewal activity of in E3 and E6. C, Relb knockdown
decreased self-renewal activity of in E3 and E6. Error bar indicates ±SD. t test was used
8
for statistical significance. * p<0.05, ** p<0.01 and *** p<0.001 significantly different
from control cells.
Figure S5. Microarray analysis of gene expression in NSCL61 and
eva1sh-expressing NSCL61 cells
A, Hierarchical clustering of NSCL61 cells and an eva1sh-expressing transformant
using the TORAY 3D-gene Mouse Oligo Chip 24k. B, Expression of the candidate
stemness-related genes in contsh- and eva1sh-expressing NSCL61 was analyzed by
RT-PCR. The expression of gapdh was used as an internal control. C, A list of the top
20 signaling pathways that were downregulated by the overexpression of eva1sh.
Figure S6. Eva1 expression level affects the NF-κB activity that is indispensable
for the proliferation of NSCL61
A, The overexpression of eva1 increased NF-κB–dependent luciferase activity in
NSCL61. B, The overexpression of eva1sh decreased NF-κB–dependent luciferase
activity in NSCL61. C, NF-κB inhibitors, CAPE (left panel) and Pterostilbene (right
9
panel), inhibited the proliferation of NSCL61. Error bar indicates ±SD. t test was used
for statistical significance. * p<0.05 and ** p<0.01 significantly different from control
cells.
Figure S7. RelB is essential for the NSCL61 proliferation and tumorigenesis
A, FLAG-tagged mouse RelA and RelB expression vectors were transfected with a
contsh, relash or relbsh expression vector into Cos7 cells. Cell extracts were harvested
2 d after transfection, and were analyzed by Western blotting using anti-FLAG and
anti-GAPDH (loading control) antibodies. B, The overexpression of relbsh decreased
NF-κB–dependent luciferase activity in NSCL61. C, The overexpression of relbsh
blocked the proliferation of NSCL61. D, Survival curve for mice transplanted with
NSCL61 cells expressing either contsh (dashed line) or relbsh (bold line). Error bar
indicates ±SD. t test was used for statistical significance. *** p<0.001 significantly
different from cells expressing control shRNA (contsh).
10
Figure S8. Relationship between relb expression and the prognosis
A, Clinical data from the cBioPortal database indicated that increased relb mRNA (red
line, Z-score>2) has correlated with a poorer prognosis, survival (left panel) and disease
free (right panel), in GBM. B, Expression data (27 microarray data) from TCGA
database indicated that relb expression significantly increased in GBM, compared with
LGG. Error bar indicates ±SD. t test was used for statistical significance.
Figure S9. Eva1+GBM cells associate with RORγT+ Th17 cells
Human GBM tissues were immunolabeled for Eva1 (green) and RORγT (red). Arrows
point the Eva1/RORγT(Th17)-double positive cells. Arrowheads point the Eva1+ cells
that associate with Th17. Nuclei were counterstained with DAPI (blue). Scale bar: 50
μm, 20 μm (insets).
Ohtsu et al,Supplementary Figure SI
0 0
4 3
uolsseJdxe
20
51
uolsseJdxe
A
02 l。eAEI
1
1
E6E3
-hGIC
E2NSC
eA!lelel:|
5
NSC NSC OPC OPC
L61 L61
1。eAEleA!lelel:|
‐3 ク一
u!pe‐gol
C
1
nl
でΦN一一喝EJOc
’」○一sse」(lxe
1。eAEI
OLIAOAAOGBMCB
rrll
AhEval
mEva1
hEval
mEva1
hEval
mEva1
hEval
mEva1
hEval
mEva1
QQCSj」o∽a″`
51
01
0.5
1
W
1
1
1
1
2
2
5
5
0
0
5
5
0
0
V
V
V
1
1
1
1
1
1
1
1
1
1
2
Ohtsu et al,Supplementary Figure S2
▼
MyGKSSTRAVLLLLGIQLTALWPエAAVEエYTSRVLEAVNGTDARLKCTFS
MyGKSPALVLPLLLSLQLTALCPTEAVEエYTSGALEAVNGTDVRLKCTFS
SFAPVGDALTVTWNFRPLDGGPEQFVFYYH工DPFQPMSGRFKDRVSWDGN
FLALA工GSACALM工工工V工VVVLF
FLAVA工GSACALM工工VV工VVVLF
V
V
V
l
5
5
0
0
10
10
15
15
2
2
0
0
0
0
0
0
0
0
SFAPVGDALTVTWNFRPRDGGREQFVFYYHMDPFRPMSGRFKDRVVWDGN
▼
£旦RYDAS工LLWKLQFDDNGTYTCQVKNPPDVDGV工GE工RLSVVHTVRFSE
PERYDVS工LLWKLQFDDNGTYTCQVKNPPDVDGLVGT工RLSVVHTVPFSE
HYRKKRWAERAHKVVE工KSKEEER
HFRKKRWADRADKAEGTKSKEEEK
一
lzg17;il・7jZj
LNQEKKVSVYLEDTD
LNQGNKVSVFVEDTD
2
2
15
15
0.4 0.08 0.0‘16 0
Ab concentration『1』9/ml)
V
V
V
じ NSC NSCL61 0PCL6-
1‐1一`j一11JI一l
-r- X C -r lx -
【III
ISr17111rm_ig!7;ilj ・ZI:¶zj
S
A contsh vector +
mEval-2XFLAG -
mEvalshRNA -
Ohtsu et al,Supplementary Figure S3
一十十
一 十 一
Eval
FLAG
GAPDH
- -
--一一
contsh vector +
hEval-2XFLAG -
hEvalshRNAI -
一十十
一 十 一
FLAG
GAPDH
-・-
四・
contsh vector +
hEval-2XFLAG -
hEvalshRNA2 -
一十十
一 十 一
FLAG
S
Ohtsu et al,Supplementary Figure S4
E6aD11 N
|leM6u一E」o`
・XCO一〇Q`○」QjEコZ
E3A60
4 2
|leM6u!E」o`
・xcO一〇〇`○」QjEコZ
EvalcontEvalcont
E6E33040
20
1
|leM
6u一EJOJ
‐XUO一〇Q`o」ΦjEコZ
0`j
一一Qヽ`’
0 0
CSO‐
6U一E」o`
・xcO一〇り`o」QjEコZ
Eval
sh2Evalshl
E6
cont
sh
Eval
sh2
Eval
shl
cont
sh
30E3
C
20
1
|leM
6u一E」o`
・xcO一〇Q`o」ΦjEコZ
0 0 0 0 0
1P11 rMsD・
|leM
6u!E」o`
・xcO一〇り`o」QjEコZ
「elbshcontshrelbshcontsh
i
ANSCL61
-3SD
NSCL61
+evafs力
+3SD
eya7
1/s&e/1e
゛S゛OQ
-
a/,が,7a3-
fley7-
ρΓdz71ブ6四,lolcM 111
abcg2-
gapa-
Ohtsu et al,Supplementary Figure S5
CNO Nelwork
Tとytll
nodes
Seed
nodes pValue
1 SPI 158 157 0.CX〕OE十〇〇
2 邱 76 75 3.140E-155
3 APjl 74 73 5.280E-151
4 ESRI 73 72 6.820E-149
5 cl/yc 69 68 1.860E-140
6 CREBI 66 65 3.840E-134
7 HIFIA 64 a3 6.150E-130
8 HNF4alpha 62 61 9.760E-123
9 C/EBPbeb 59 58 1.920E-119
10 cJun 56 55 3.680E-113
11 RelA 53 52 6.910E-107
12 STAT3 52 51 8.480E-105
13 STATI 49 48 1.550E�8
14 E2FI 48 47 1.880E96
15 NFkB 43 42 4.8〔X⊃E名6
16 PU.1 43 42 4.8〔X⊃E名6
17 SRF 42 41 5.740E名4
18 c-Fcx5 40 39 8.160E名0
19 EGRI 39 38 9.6g⊃E78
2⊃ Andr)genEce㈲「 39 38 9.6g⊃E78
--」」
四回
A
C
4!A!pe」30Eo」一
IDNQ CsD・
120
8 6 40 2
4!|!qe!A%
None NF-KB API SPI
120
5
9 CQ CO‐
4!A!lc)e」30EO」一
120
0 0 0
g CPlt
4!|!qe!A%
0 0
肖/`
Ohtsu et al,Supplementary Figure S6
None NF-KB API SPI
Tralx申tk)nfactorbildilg sies
20 40 60 80 100
lnhibitor(1j9/ml)
Tralxr4)tionfi・ctorbindilg sies
1001nhibitor(1JM)
一!
LI I
O
A 4盈QOQ
FLAG(RelA)|-・
GAPDH
4!A!13e」SOEo」一
100
0
″D
一gンーヽ’」コ∽g
●
l/se/aj
&/sq/aj ■㎜
,j‾
---
Ohtsu et al,Supplementary Figure S7
FLAG(RelB)
C
GAPDH
60 40 20
slle3+コで」9%
4jQou
9se/aj
&lsq/ej
‾㎜4、
rellsh
40
contsh |「isJF;7Sic(iltsh
□cont ■NF-KB
relash
10 20
Days
relbsh
31
rrll
A100
60 40 20
一eA!AJns%
でΦN=SE」OCN㎝OJ
2
1
1
-2
20
LGG
Median survival
-7.59 M(9) -13.3 M(136)
pvalue : 0.0519
eeJJ
100
60 40
esees!p 20
Ohtsu et al,Supplementary Figure S8
Median disease free
-2.89 M(5)
-7M(108)
pvalue : 0.0033
20 40 60
Months
40 60
Months
ρ:2.51E-4-
GBM
ヒ!
U5-Ug
S
●‘鼠'
?.・
、7 t
″
.s.
i
〃==========
| |
l.,
r’I
|……….j
rl
1
●
1...こ'.....j
●・
●・
Ohtsu et al,Supplementary Figure S9
4
→
→
4
4
▼
▲I-
哺
▼
↑ 4
-・▼
・哺
←
-▼
・哺
←
▼
・’→烏
|=g゛7;i!M曼lialj・Z1211j
し
↑ 4
一・
-・▼。
4
←㎜