Eva1 Maintains the Stem-like Character of Glioblastoma ... · Title Eva1 Maintains the Stem-like...

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Instructions for use Title Eva1 Maintains the Stem-like Character of Glioblastoma-Initiating Cells by Activating the Noncanonical NF-κB Signaling Pathway Author(s) Ohtsu, Naoki; Nakatani, Yuka; Yamashita, Daisuke; Ohue, Shiro; Ohnishi, Takanori; Kondo, Toru Citation Cancer research, 76(1), 171-181 https://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

Transcript of Eva1 Maintains the Stem-like Character of Glioblastoma ... · Title Eva1 Maintains the Stem-like...

Page 1: Eva1 Maintains the Stem-like Character of Glioblastoma ... · Title Eva1 Maintains the Stem-like Character of Glioblastoma-Initiating Cells by Activating the Noncanonical NF-κB Signaling

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

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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

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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'.

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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).

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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,

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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.

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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

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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

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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

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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).

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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).

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Ohtsu et al,Supplementary Figure SI

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