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