Supplementary Figure Legends Figure S1. fileblotting analysis of PTEN protein levels was carried out...
Transcript of Supplementary Figure Legends Figure S1. fileblotting analysis of PTEN protein levels was carried out...
Supplementary Figure Legends
Figure S1. HDAC inhibitors activate caspase-3 in synovial sarcoma cells. Western blot
analysis was performed to assess the levels of full and cleaved caspase-3 in SYO-1 and
FUJI cells treated with 10 nM romidepsin, 10 M MS-275, 1 M PXD101, or DMSO for
16 hr. Actin is used as a loading control.
Figure S2. The protein level of EGR1 in human embryonic kidney HEK293 and synovial
sarcoma SYO-1 and FUJI cells. Western blots against EGR1 were done using HEK293,
SYO-1, and FUJI cells; the fusion oncoprotein SS18-SSX2 was detected as a marker for
synovial sarcoma. Actin is a loading control.
Figure S3. EGR1 is specifically induced by romidepsin in stable HEK293 cells
expressing the SS18-SSX2 oncoprotein. RT-qPCR analysis of EGR1 transcription was
done using stable HEK293 cell lines expressing Myc-tagged His, SS18, or SS18-SSX2
with 9 hr romidepsin treatment. 18S rRNA is applied as an internal control.
Figure S4. EGR1 knockdown reduces FUJI cell killing by romidepsin treatment. (a)
EGR1 mRNA levels were determined by RT-qPCR using control or EGR1-knockdown
FUJI cells. (b) Cellomics analysis of FUJI cells transfected with control or EGR1 siRNA
in the 48-hr treatment of romidepsin or DMSO. Data are representative of three
independent experiments; bars, 95% CI.
Figure S5. Knockdown of EGR1 in the synovial sarcoma cell lines SYO-1. SYO-1 cells
were transduced with or without sh-Control or sh-EGR1 lentiviral construct as described
in Materials and Methods. EGR1 and actin protein levels were assessed by western blot
analysis 6 hr after romidepsin addition.
Figure S6. EGR1 knockdown reduces the sensitivity of SYO-1 cells to MS-275-induced
apoptosis. (a) RT-qPCR analysis of EGR1 transcripts in sh-Control and sh-EGR1 SYO-1
cells treated with 10 M MS-275 or 0.1% DMSO for 12 hr. (b) Average percentage of
control or EGR1-knockdown cells positive for PI after 32 hr post addition of MS-275 or
DMSO.
Figure S7. Romidepsin directly induces EGR1, but not PTEN, in synovial sarcoma cells.
(a) RT-qPCR analysis of EGR1 mRNA levels was performed using SYO-1 cells
incubated with or without cycloheximide (CHX) for 30 min followed by romidepsin
treatment. (b) RT-qPCR was done in the same cultures as described above.
Figure S8. The effect of EGR1 and PTEN on AKT phosphorylation in synovial sarcoma
cells. (a) The levels of p473-AKT and total AKT were assessed by western blots using
control, EGR1-, and PTEN-expressing SYO-1 cells. The transfection efficiency was also
examined by western blots, and -Actin was indicated as a loading control. (b) Western
blotting analysis of PTEN protein levels was carried out on SYO-1 samples transfected
with the indicated siRNAs and following treatment with romidepsin.
Figure S1. Le Su et al.
Caspase-3
cleavedCaspase-317
2836
5572
130
0.1%
DM
SO
1uM
PX
D10
1
10n
M F
K22
8
10u
M M
S-2
75
(kDa)
Actin
SYO-1
0.1%
DM
SO
1uM
PX
D10
1
10n
M F
K22
8
10u
M M
S-2
75
FUJI
55
72
95
(kDa)
130
EGR1
SYT-SSX2
Actin
HEK293
SYO-1
FUJI
Figure S2. Le Su et al.
180HEK_Ctrl
HEK_SS18
HEK_SS18-SSX2160
140
120
80
60
40
20
100
00 hr FK2286 hr 9 hr
% E
GR
1 G
ene
Exp
ress
ion
Figure S3. Le Su et al.
Figure S4. Le Su et al.
ANRis lrtC
ANRis1
GRE
a b
0102030405060708090
100110120
02
46
810
1214
1618
2022
2426
2830
3234
3638
4042
4446
48(hr)
noisser
pxE
E
GR
1
%
FUJI
si-EGR1 + FK228
si-Ctrl
si-EGR1
si-Ctrl + FK228
0.30
0.60.91.21.51.82.12.42.73.03.3
PI p
osi
tive
cel
ls (
10
)
3
Paren
tal
sh-C
trl
sh-E
gr1-1
sh-E
gr1-2
Paren
tal
DMSO
FK228
72
130
(kDa)
95
250
55
36
28
17
EGR1
Actin
Figure S5. Le Su et al.
Figure S6. Le Su et al.
b
0
10
20
30
40
50
60
70
80
90
100
0 8 16 24 32 (hr)
% C
ell D
eath
(P
I+ c
ells
)
sh-EGR1 + MS-275
sh-Ctrl
sh-EGR1
sh-Ctrl + MS-275
a
0
100
200
300
400
500
600
0 hr 3 hr 6 hr 12 hr
% E
GR
1 G
ene
Exp
ress
ion sh-Ctrl
sh-EGR1
MS-275 Treatment
Figure S7. Le Su et al.
0
50
100
150
200
250
300
350
400
450
0
50
100
150
200
250
300
350
% E
GR
1 G
ene
Exp
ress
ion
% P
TE
N G
ene
Exp
ress
ion
a b
FK228 FK228
CHX
DMSO
Vec
tor
EG
R1
PT
EN
PTEN
β-Actin
EGR1
total AKT
p473-AKT
cDNA:
a b
Figure S8. Le Su et al.
72
55
36
PTEN
FK228 (hr)
β-Actin
(kDa)0 6 12 0 6 12 0 6 12
si-EGR1 si-PTENsi-CTRL