supplemental figure 1dm5migu4zj3pb.cloudfront.net/manuscripts/40000/... · supplemental figure 4 0...
Post on 23-Sep-2020
1 Views
Preview:
TRANSCRIPT
20 nm
a
Supplemental figure 1
b
0
20
40
60
80
100
120
140
160
Medium EL4 TS/A CT26
** *
Ace
tylc
ho
line
este
rase
act
ivit
y (a
.u.)
Figure S1
a : Picture from the ultracentrifuged pellet from culture supernatant of EL4.
b: Acetylcholine esterase activity in ultracentrifuged fraction (TDE).
Supplemental figure 2
Figure S2 :Stat3 activation in purified MDSCs
Purified splenic MDSCs from naïve mice were treated with TCS or TDSF or TDE. pStat3
expression was determined by FACS analysis. Data are Mean ± s.d. fluorescence intensity (n=3)
Norm
aliz
ed p
Sta
t3 M
FI
0
50
100
150
200
250
300
350
400
TSA EL4 CT26 TSA EL4 CT26 TSA EL4 CT26
CM Tumor cell supernatant TDSF TDE
* * * * * *
TCS
Supplemental figure 3
0
10
20
30
40
50
60
70
80
90
CM TDSF TDSF + aGM-CSF GM-CSF
% G
r1+
CD
11b c
ells
* *
Figure S3. Expansion of MDSCs by GM-CSF
Bone marrow from naïve mice were cultured 3 days in complete medium alone or with TDSF or TDSF
+ blocking anti GM-CSF or rGM-CSF . The percentage of Gr1+ CD11b+ precursor cells ± s.d. was
determined by flow cytometry.
a GM-CSF
Supplemental figure 4
0
0,2
0,4
0,6
0,8
1
1,2
1,4
0 2 4 6
p6
5 O
.D.
Time (h)
0
0,2
0,4
0,6
0,8
1
1,2
1,4
Co TDSF GM-CSF
p6
5 O
.D.
TDE exposure
Figure S4. NFkB p65 transcriptional activition by TDE and TDSF
(Left panel) MDSC clone were stimulated with TDE for 2h, 4h or 6h . Then NFkB p65 transcriptional
activity was determined with TransAm assay.
(Right panel) MDSC clone were stimulated with TDSF or GM-CSF for 2h. Then NFkB p65
transcriptional activity was determined with TransAm assay.
*
*
*
Supplemental figure 5
pStat3
Naive mice Tumor bearing mice
IgLy6Cint Ly6G+Ly6Chi Ly6G-
Figure S5 . pStat3 expression in MDSCs Subpopulations
MDSC from tumor-bearing mice were analysed for Ly6C Ly6G CD11b and pStat3 expression by
cytometry.
Monocytic MDSC were characterized by Ly6ChighLy6G-CD11b+; granulocytic MDSC by Ly6Cintermediate
Ly6G+ CD11b+.
Supplemental figure 6
a
b
0
50
100
150
SCF PGE2 IL-6 IL-10
EL4
TS/A
CT26
Figure S6. Cytokines production by tumor cells and TDE
A. Tumor cell supernatant SCF, PGE2, IL-10 and IL-6 were determined by ELISA.
B. PGE2 levels in TDE were determined by ELISA.
pg
/ml
0
1000
2000
3000
4000
5000
1 2 3TS/A CT26 EL4
5
4
3
2
1
0
ng
/µg
Supplementary figure 7
Il-1b
pg
/ml
0
50
100
150
TS/A EL4 CT26 TS/A EL4 CT26 3T3
CM UC Medium Exosome
TNF-a
pg
/ml
a
b
IL-1
0 p
g/m
l
c
d
VEG
F p
g/m
l
0
50
100
150
0
50
100
150
0
50
100
150
TDSF TDE
Figure S7. Cytokines production by MDSCs.
Purified MDSC from naïve mice were treated 24h with complete conditioned medium (CM) or
soluble fraction (TDSF) or exosome (TDE) obtained from CT26, TS/A, EL4 and 3T3 cell cultures
before and after ultracentrifugation (UC). IL-1b (a), TNF-a (b), IL-10 (c) and VEGF (d)
concentrations were determined by ELISA.
Supplementary figure 8
0
200
400
600
800
WT TLR2 TLR4 Myd88 Trif
Untreated
LPS
PolyICIL-6
pg
/ml
a
0
100
200
300
400
500
WT TLR2 TLR4 Myd TRIF
untreated
IL-6
No
rmal
ized
Pst
at3
MFI
pSt
at3
MFI
WT TLR2-/- TLR4-/- MyD88-/- Trif-/-
WT TLR2-/- TLR4-/- MyD88-/- Trif-/-
b
Figure S8. IL-6 production and pStat3 expression by MDSCs from wild type and TLR
deficient mice
a. MDSCs from wild-type or TLR2-, TLR4-, MyD88- or TRIF-deficient tumor-free mice were
cultured for 24h with PBS alone or supplemented with LPS (10 ng/mL) or Poly(I:C) (50μg/mL),
then IL-6 concentrations were determined by ELISA in the supernatant.
b. MDSCs from wild-type or TLR2-, TLR4-, MyD88- or TRIF-deficient tumor-free mice were
cultured for 24h with PBS alone or supplemented with rIL-6 (10ng/mL). pStat3 expression was
determined by FACS analysis on MDSCs gated cells. MFI = mean fluorescence intensity ± s.d.
Supplemental figure 9
tsg101
Grp94
Hsp72
Exo
som
es
Mic
rop
arti
cles
EL4
Figure S9. Expression of Hsp72, Tsg101 and Grp72 in exosome and other microparticules
EL4- exosome were separated from other microparticules by ultracentrifugation on 30% sucrose/D2O.
Both fractions were blotted for tsg101 (exosomal protein), Grp94 (endoplasmic reticulum protein) and
Hsp72.
Supplemental figure 10
0
100
200
300
400
500
600
700
untreated rHsp72 Pam3CSK4 Pam3CSK4+ anti Hsp72
Pg/mL
IL-6
TNF-a
Figure S10. IL-6 and TNF-a production by MDSCs
Naïve MDSCs were stimulated 24h with rHsp72 or PAM3CSK4 or PAM3CSK4 plus a blocking anti
Hsp72. IL-6 and TNF-a in the supernatant were determined by ELISA.
Supplemental figure 11
rHsp72pErk
Erk
pStat3
Stat3rHsp72
0 5 15 30 60Time (min) 0 2 4 6Time (h)
0
0,4
0,8
1,2
1,6
0 2 4 6
p6
5 O
.D.
Time (h)
rHsp72
0
0,2
0,4
0,6
0 2 4 6
pSt
at3
O.D
.
Time (h)
rHsp72
Figure S11. Erk ,Stat3 and NFkB activation by Hsp72 in MDSCs
MDSC clone were stimulated with rHsp72, pERK (a) and pStat3 (b) expression was determined western
blotting.
MDSC clone were stimulated with rHsp72, then NFkB p65 (c) and Stat3 (d) transcriptional activity was
determined with TransAm assay.
a b
c d
Supplemental figure 12
1
0.8
0.6
0.4
0.2
Hsp72
b-actin
Mock H95 H96 H97 H98 H99
Fo
ld d
ecre
ase
0
b-actin
Hsp72
00.20.40.60.8
1
Fold
decre
ase
Mock H96 H97
TDE
Figure S12. Hsp72 expression in CT26 clones transfected with ShRNA targeting Hsp72
Hsp72 expression was determined by western blotting in shRNA Mock CT26, and in 5 different shRNA
Hsp72 CT26 clones. Hsp72 expression was also determined in TDE from shRNA Mock CT26 and shRNA
Hsp72 CT26 clones H96 and H97.
Supplementary figure 13
0
500
1000
1500
2000
2500
0 5 10 15 20
0
500
1000
1500
2000
2500
0 5 10 15 20
Wild-Type NudeTu
mo
r vo
lum
e (m
m3)
Tum
or
volu
me
(mm
3)
TS/A
EL4
CT26
PBS
CTX
0
1000
2000
3000
4000
5000
0 6 9 12 14 17 20
Tum
or
volu
me
(mm
3)
0
1000
2000
3000
4000
5000
0 6 9 12 14 17 20
Tum
or
volu
me
(mm
3)
0
200
400
600
1 6 11
0
200
400
600
1 2 3 4 5 6 7 8 9 10 110 10 20
1500
3000
4500
* * *
** **
*
** * *
0 10 20
Tum
or
volu
me
(mm
3)
Figure S13. Tumor growth in wild type and n udemice after cyclophosphamide treatment.
Wild-type and nude mice were subcutaneously injected with 1.106 TS/A, EL4 or CT26 tumor cells. 10
days later, mice were injected with PBS or cyclophosphamide (CTX) (100 mg/kg). Tumor volume was
determined using a caliper.
4500
1500
3000
Tum
or
volu
me
(mm
3)
H23
b-actin
Hsc73
Hsp72
Supplemental figure 14
Figure S14.
Hsc73 and Hsp72 expression in H23 and H23 TDE was determined by western blotting.
top related