Acancerdiscovery.aacrjournals.org/content/suppl/2015/01/10/2159...analysis and using the...
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B A WT T286A 0 2 4 6 WBC (K/ul) ** 0 50 100 Spleen (mg) ** WT T286A 0 5 10 RBC (M/ul) WT T286A 0 500 1000 PLT (K/ul) ** WT T286A 0 10 20 30 40 50 Thymus (mg) WT T286A ** ** Supplemental Figure 1 C 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 0 20 40 60 80 10 0 10 1 10 2 10 3 10 4 0 30 60 90 120 CD11b Gr-1 CD4 CD8 B220 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 0 200 400 600 800 1000 NGFR SSC D M Vβ 1 Vβ 2 Vβ 3 Vβ 4 Vβ 5 Vβ 6 Vβ 7 Vβ 8.1 Vβ 8.2 Vβ 8.3 Vβ 9 Vβ 10 Vβ 11 Vβ 12 Vβ 13 Vβ 14 Vβ 15 Vβ 16 Vβ 17 Vβ 18 Vβ 19 Vβ 20 (-) (+) WT Tumor 1 Tumor 2 Tumor 5 Tumor 3 Tumor 4 Tumor 6 Tumor 7 100 bp 200 bp 100 bp 200 bp 100 bp 200 bp 100 bp 200 bp 100 bp 200 bp 100 bp 200 bp 100 bp 200 bp 100 bp 200 bp
Transcript of Acancerdiscovery.aacrjournals.org/content/suppl/2015/01/10/2159...analysis and using the...
B
A
WT T286A 0
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Supplemental Figure 1
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Supplemental Figure 2
D E
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CD4 0 102 103 104 105
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f cel
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8ICN1+PRMT5 ICN1 A
Supplemental Figure 3
E
meR-p53 p53 β-Actin
PRMT5
-
AF9
AF9+PRMT5
myc-PRMT5
F
myc-PRMT5Δ
AF9
AF9
+PR
MT5∆
AF9
1 2 3 4 Mouse #
Before BMT ~2 months after BMT A
F9+P
RM
T5∆
PRMT5 (endo)
β-Actin
G
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2 round 3 round 4 round 5 round
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ony
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C
GFP
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FR
CD11b
Gr-
1
AF9+PRMT5 AF9
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Supplemental Figure 4
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GFP
SSA
CD4
CD
8
2.43 20.2 20.3 36.2 29.9 87.5 3.66
P=0.015
A.
D.
L
S
T
T286A/p53-/-
GFP/p53-/-
T286A/p53-/- WT D1/p53-/- GFP/p53-/-
B.
0 102
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P=0.0002 F.
E. Liver Spleen Thymus
Lung Kidney
Supplemental Figure 5
GFP
mCherry
Merged
T286A+PRMT5
T286A+PRMT5
T286A+p53DN
T286A+p53DN
B
p53
PRMT5
PRMT5
meR-p53
-
+ +
-
+
+
-
-
- -
++
WT
-
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+ + RK -
-
D
DAPI
p53
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meR-53
H1299
p53 WT
p53 RK
C
myc-PRMT5 Endo-PRMT5
β-Actin
Cyclin D1
A
p53
p53D
N
PR
MT5
P
RM
T5Δ
-
p53D
N
PR
MT5
P
RM
T5Δ
-
p53D
N
PR
MT5
P
RM
T5Δ
-
D1
T286
A
-
PRMT5
3H Me p53
p53 (Coomassie)
p53 WT p53 RK B
P∆ P∆ P P - -
A - PRMT5 -
PRMT5
3H Me p53
p53 (Coomassie)
WT WT RK - RK
T286A/CDK4 - p53 WT
PRMT5
3H Me p53
p53 (Coomassie)
MEP50
p53 RK D PM - PA PD PM - PA PD
C PRMT5
32P p-Rb Rb (Coomassie)
D1T286A/CDK4
p53(Coomassie)
3H Me p53
PRMT5
- + - + + + +
+ p53
IgG
PRMT5
DAPI
MeR-p53 D1T286A
Merged
ç ç ç
ç ç
ëëë
ííí
F E
DAPI Merged
MeR-p53 D1T286A
ìì
çç
ë
é
Supplemental Figure 6
T286A/CDK4 -
Supplemental Figure 7
Apaf1 Cdkn1a Bax Pmaip1 A
B Apaf1 Cdkn1a Bax Pmaip1
* *
0
0.2
0.4
0.6
0.8
1
1.2
% in
put
*
0 0.5
1 1.5
2 2.5
3 3.5
% in
put
0
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% in
put
0
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% in
put
*
0 0.5
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% in
put *
*
0
5
10
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20
25
% in
put
0
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10
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20
25
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% in
put
* *
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
% in
put
A
C
Supplemental Figure 8
PRMT5 high
PRMT5 low
Total cases
D1 high 34 21 55
D1 low 9 8 17
Total cases
43 29 72
D
B
Normal ESCC
Normal (13-‐12521 )
MCL LN (10-‐0027972 )
IgG control
MCL LN (11-‐0025536 )
Normal (13-‐12521 )
MCL LN (10-‐0027972 )
IgG control
MCL LN (11-‐0025536 )
SUPPLEMENTAL INFORMATION
SUPPLEMENTAL EXPERIMENT PROCEDURES
T Cell Receptor (TCR) clonality analysis
TCR clonality was assessed using receptor-specific antibodies followed by FACS
analysis and using the SuperTCRExpress Mouse T Cell Recptor (TCR) Vβ Repertoire
Clonality Detecting Kit (BioMed Immunotech, Tampa, FL) according to manufacturer’s
instructions.
In Vitro Methyltransferase Assay
In vitro methyltransferase assay was carried out as described (7). In brief, for assessment
of PRMT5 methyltransferase activity, PRMT5 or PRMT5/MEP50 complexes were
immunopurified from transfected 293T cells. To generate PRMT5 or PRMT5/MEP50
complexes, myc-tagged alleles were expressed in 293T cells and immune-purified using
anti-c-myc-Agarose beads (Sigma). Equimolar PRMT5-MEP50 was confirmed by
immunoblot with the 9E10 antibody and the absence of endogenous MEP50 was assessed
by immunoblot with a MEP50-specific antiserum. Beads were washed in Tween-20
buffer followed by methyltransferase buffer (15mM HEPES, pH7.9), 100mM KCl, 5mM
MgCl2, 20% Glycerol, 1mM EDTA, 0.25mM DTT and 0.5mM PMSF). The methylation
reaction included PRMT5 immune complexes on beads, recombinant p53 (1µg) and 2.75
µCi S-adenosyl-L-(methyl-3H)methionine (Amersham Pharmacia) in a total volume of 25
µl for 1.5 hours at 30 oC. The reaction mixture was resolved on a SDS-polyacrylamide
gel, and modified p53 was detected by fluorography. For the coupled kinase
assay/methyltransferase assay, cyclin complexes were purified from Sf9 cells as
described in (17). They were mixed with PRMT5/MEP50 complexes immobilized on
beads, for 30 minutes with ATP at 30°C. Following extensive washes, phosphorylated
PRMT5/MEP50 complexes were then used in methyltransferase assays as described
above.
Antibodies
Antibodies for flow cytometry analysis: BD Pharmingen: NGFR (CD271 Alexa fluor 647,
C40-1457), CD4 (PerCP-CyTM 5.5, RM4-5), CD8 (PE, 53-6.7) and B220 (PerCP- CyTM 7,
RA3-6B2). BD Horizon: Lineage antibody cocktail (V450). eBioscience : Sca-1 (PE,
D7), c-kit (PE-Cy7, 2B8), CD127 (APC-eFluro 780, A7R34), CD25 (PE, PC61.5),
CD69 (APC, H1.2F3), CD44 (PE, IM7), CD62L (APC, MEL-14) , CD3 (PE-CY5.5,
145-2C11) , CD4 (eFluor® 450, GK1.5 ), CD8 (APC-eFluor® 780, 53-6.7), TCR β
(APC-eFluor 780, H57-597), CD16/CD32 (93), Fixable Viability Dye (eFluor® 506), V
beta 2 TCR (PE), V beta 3 TCR (Biotin), V beta 4 TCR (PE), V beta 6 TCR (PE), V beta
7 TCR (PE), V beta 8 TCR (PE), V beta 10 TCR (PE), V beta 11 TCR (PE), V beta 12
TCR (Biotin), V beta 14 TCR (Biotin) and PE anti-biotin, and DAPI (Sigma). For
immunoblot analysis: cyclin D1 (D1-17-13G), PRMT5 (PRMT5-21, Santa Cruz), MEP50
(A301-562A, Bethyl), p53 (DO-1 and Pab 240 Santa Cruz), ß-actin (AC-15, Sigma) and
GAPDH (14C10, Cell Signaling Technology). For immunohistochemistry: PRMT5
(ab31751, Abcam). For CHIP assay: p53 (FL-393, Santa Cruz), E2F-1 (C-20, Santa
Cruz), H4R3 (ab5823, Abcam).
SUPPLEMENTAL TABLE
Table S1. T cell receptor (TCR) Vβ repertoire usage in CD3+CD4+ T cells from tumor-
bearing and non tumor-bearing mice.
Percentage of Vβ in CD3+CD4+ cells
Naive Tumor 1 Tumor 2 Tumor 3 Tumor 4 Tumor 5 Tumor 6 Tumor 7
Vβ 2 2 0 0 0 0 0 0 0
Vβ 3 4 0 10 0 13 3 0 0
Vβ 4 7.5 0 0 0 0 0 0 0
Vβ 6 8 0 0 5 0 0 3 0
Vβ 7 2 0 0 0 0 0 0 0
Vβ 8 22 0 0 0 0 0 0 0
Vβ 10 4 0 0 0 0 0 0 0
Vβ 11 4 0 0 0 0 0 0 0
Vβ 12 4 1 0 0 0 0 0 0
Vβ 14 9 95 0 0 0 0 0 0
Total TCR 66 96 10 5 13 3 3 0
SUPPLEMENTAL FIGURE LENGENDS
Figure S1. Pancytopenia in D1T286A-reconstituted mice, immnuphenotype of
PRMT5-reconstituted mice and TCR clonality of D1T286A+PRMT5 mice. (A)
Complete blood counts for D1T286A (T286A) mice. WBC, white blood cell; RBC, red
blood cell; PLT, platelet.**p<0.01. (B) Spleen and thymus weight for D1T286A mice.
**p<0.01. (C) FACS of bone marrow derived single cell suspensions of PRMT5 positive
and negative cells in PRMT5 reconstituted mice. (D) SuperTCRExpressTM Mouse T Cell
Receptor (TCR) Vβ Repertoire Clonality Detecting Kit was used to assess TCR clonality
from tumor-bearing (tumor 1~7) and non tumor-bearing (WT) mice. M: 25 bp DNA
marker; (-):negative control, (+):positive control from a monoclonal T cell line.
Figure S2 Phenotype of the D1T286A+PRMT5 lymphoma and the D1T286A and
PRMT5 triggered lymphoma is 100% transplantable. (A-C) Splenocytes from
D1T286A (T286A) +PRMT5 mice were assessed by flow cytometry. (A) Expression of
the CD3, CD4, TCR Vβ and CD8. (B) Expression of CD25/CD69 in the CD4+CD3+
population. (C) Expression of CD44/CD62L in the CD4+CD3+ population. (D and E)
Splenocytes from WT and T286A+PRMT5 mice stimulated with 30 µg/ml
lipopolysaccharides were treated with 100 ng/ml Colcemid for 2 h. Metaphase spreads
were prepared and stained with Giemsa to visualize chromosome alterations. (D)
Quantification of the average number of chromatid breaks per cell; **p<0.01 (E)
Quantification of the chromosome number (n > 40) per cell for WT (red) and T286A
+PRMT5 (blue) mice splenocytes. (F) Kaplan-Meier survival curve of secondary and
tertiary (red) transplants. Sublethally irritated mice (600 rads) received different numbers
of bone marrow or spleen cells from primary or secondary lymphomatous mice. (G)
Immunophenotype of D1T286A and PRMT5-induced lymphoma after secondary
transplant. Cells from bone marrow and spleen were assessed by flow cytometry. The
CD4 and CD8 profile of the GFP+ and NGFR+ population is shown. (G) FACS of
secondary tumors of the indicated genotypes (LSK, Lin-Sca-1+c-Kit+) from bone marrow
of WT and TA+PRMT5 mice.
Figure S3. PRMT5 is required for leukemia driven by MLL-AF9. Immunophenotype
of tumors that developed in ICN1 (A), (B), MLL-AF9 (C) reconstituted mice. (D-F) 5-
FU–treated bone marrow cells were transduced with MigR1 (-), MigR1- MLL-AF9
(AF9) and tNGFR-myc-PRMT5 retroviruses as indicated and transplanted into recipient
mice. (D) Kaplan-Meier survival curves. (E) Western blots of PRMT5, meR-p53 and p53
in tumor-bearing spleen of the recipient mice. (F) Western blots of PRMT5 in bone
marrow cells before transplantation (left) and tumor-bearing spleen ~2 months post-
transplant (right). (G) 5-FU–treated bone marrow cells were transduced with MigR1-
MLL-AF9 (AF9) and tNGFR-myc-PRMT5∆ retroviruses and plated into methycellulose.
Cells were replated after 7 days culture and colonies were enumerated after another 7
days; * p< 0.05, ** p< 0.01.
Figure S4. Cyclin D1T286A triggers T/B-cell lymphoma in p53 dependent way. (A)
FACS analysis of bone marrow in D1T286A (T286A)/p53-/- chimera mice with T- cell
lymphoma. (B) Quantification of A. (C) FACS analysis and quantification of bone
marrow in D1T286A/p53-/- chimera mice with B-cell lymphoma. (D) Representative
photographs of splenomegaly, thymumegaly and enlarged liver in D1T286A p53-/- mice
compared to their age- matched littermates of GFP p53-/- mice. Thymus (T), Liver (L),
Spleen (S). (E) Histology of the spleen, liver, thymus, lung and kidney of tumor burdened
mice of the indicated genotype. Scale bar, 1,000µm. (F) FACS of bone marrow derived
single cell suspensions in D1T286A+p53DN reconstituted mice.
Figure S5. Confirmation of Cyclin D1T286A/PRMT5 expression and p53me2
antibody. (A) 5- FU– treated bone marrow cells were transduced with MigR1-D1T286A
(TA), tNGFR-myc- PRMT5, tNGFR-myc-PRMT5Δ or tNGFR-p53DN retroviruses as
indicated. 48 hours later, cells were subjected for western blot. (B) 5-FU– treated bone
marrow cells were transduced with MigR1-D1T286A (T286A), mCherry-PRMT5 and/or
mCherry-p53DN retroviruses. Cells were plated into methycellulose media and
GFP+/mCherry+ colonies were picked up and replated 7 days later. Colonies were
enumerated and photographed after another 7 days. Scale bar, 800 µm. (C) p53 null
H1299 cells were infected with pBabe-p53 WT or pBabe-p53RK mutant retroviruses for
48 hour. Then cells were stained with p53 (DO-1) antibody and a p53me2 antibody
generated against a peptide containing symmetrically dimethylated arginines 333,335,337
(meR-p53). (D) PRMT5 was immunoprecipitated from 293T cells transfected with
pcDNA3-myc-PRMT5 and the methyltransferase activity was assessed using S-
adenosylmethionone and recombinant WT or RK mutant of p53. p53 methylation was
assessed using the meR-p53 antibody.
Figure S6: Cyclin D1T286A/CDK4 phosphorylation of MEP50 increases PRMT5-
dependent methylation of p53. (A) myc-tagged PRMT5 was immunopurified from
293T cells transfected with pcDNA3-myc-PRMT5 using myc-agarose and the
methyltransferase activity was assessed using 3H-SAM and recombinant WT or RK p53.
(B) myc-tagged wild-type PRMT5 (P) or PRMT5∆ (P∆) was immunoprecipitated from
293T cells and methyltransferase activity was assessed using the indicated recombinant
substrates. (C) Purified cyclinD1 T286A/CDK4 complexes were incubated with
immunopurified PRMT5/MEP50 in kinase buffer with ATP to permit phosphorylation.
Following extensive washes, PRMT5/MEP50 methyltransferase activity was assessed.
(D) 293T cells were transfected with PRMT5 (P) along with either wild-type MEP50 (M)
or MEP50T5A (A) or MEP50T5D (D) mutants. PRMT5/MEP50 complexes were
purified using myc-agarose and PRMT5 methyltransferase assay was assessed as in C.
(E) NIH3T3 cells were transfected with MigR1-D1T286A and subjected to
immunofluorescence with meR-p53 antibody. Single arrow: untransfected cell; double
arrows: cells transfected with D1T286A. Scale bar, 20 µm. (F) NIH3T3 cells were co-
transfected with MigR1-D1T286A and mCherry-PRMT5 and stained with meR-p53
antibody. Single arrow: untransfected cells; double arrows: cells transfected with PRMT5
only; triple arrows: cells co-transfected with PRMT5 and D1T286A. Scale bar, 20µm.
Figure S7. Effect of PRMT5 and D1T286A on the Apaf1, Cdkn1a, Bax and Pmaip1
promoters. 5-FU–treated bone marrow cells were transduced with MigR1 and tNGFR
vectors (-), cyclin D1T286A (T286A) or/and PRMT5 and transplanted into lethally
irradiated recipient mice. Nine days after transplantation, GFP and NGFR double positive
bone marrow cells were sorted. ChIP was performed using IgG (red), E2F-1(C-20) (blue,
A) or 3 dimethyl specific H4R3 (blue, B) antibodies; *p<0.05.
Figure S8. PRMT5 and/or H4R3 are elevated in primary human cancers. (A)
Immunohistochemical staining of IgG (top left) and PRMT5 (top right and bottom) in
primary human mantle cell lymphoma samples (MCL, case numbers indicated in
parentheses). Scale bar, 800 µm. (B) Immunohistochemical staining of IgG (top left) and
H4R3 (top right and bottom) in primary human mantle cell lymphoma samples. Scale bar,
800 µm. (C) Represent immunohistochemical staining of PRMT5 in primary human
ESCC samples. Left: Normal esophageal squamous epithelium, Right: Esophageal SCC.
Scale bar, 800 µm, Note that the PRMT5 immunohistochemical staining is both
cytoplasmic and nuclei. (D) Quantification of B.
![IMMUNOGLOBULINE E T CELL RECEPTOR T. Strachan e A.P. … · B cell antigen receptor tetramero [ IgH 2 + IgL 2 (Ig oppure Ig )] T cell receptor (TCR) eterodimero TCR /TCR TCR /TCR](https://static.fdocuments.us/doc/165x107/5c017b5c09d3f26f1e8cc6a0/immunoglobuline-e-t-cell-receptor-t-strachan-e-ap-b-cell-antigen-receptor.jpg)
