1. A B
Pax8
CK7
CA125
HE4 HE
xenograft
tumor
primary
#20
bright
phase HE C D E
Nanog
Sox2
ALDH1A1
CK7
Actin #5
#20
diff.
spheroid
xenograft
tumor
primary #5
ALDH1A1
Nanog
Sox2
CK7
#20
spheroid
diff.
***
*** *
***
relative RNA levels
Fig. S1. Spheroid cells derived from human ovarian cancer show characteristics as CSCs
a. spheroid picture (HE/phase contrast), SOC-20
b. xenograft & primary (HE), SOC-4, scale bar 50um
c. xenograft & primary (HE), SOC-5, scale bar 50um
d.. western, SOC-5, 20
e.. real time, SOC-20
Ishiguro et al. Supplementary Figure S1

2. A B C #2 #5
14.8%
69.9%
50 mM
DEAB + -
27.3%
31.4%
90.1%
0.0%
86.6%
ALDH activity
0.4%
31.9%
anti-CD133
IgG
CD133
ALDHlow
relative caspase activity
ALDHhigh
*** D E F G
ALDH1A1
SOX2
Oct-3/4
Nanog
Actin
CD133
CD133low
CD133high
ALDH
CD133
High High Low Low
High Low High Low
relative caspase activity
relative cell growth
(days)
CD133 high
CD133low
CD133low
CD133high
n.s.
fig. S2 High levels of ALDH expression is maintained by ROCK inhibition and associated with proliferation of ovarian CSCs
a. Growth w or w/o Y insulin (SOC-2,4,5)
b. ALDH, SOX2, Nanog Western (SOC-2,4,5)
c. aldeflour
d. ALDH, SOX2, cd44 Western (SOC-2,4,5) w or w/o Y27632
e. ALDH immunostaing
n.s.
Ishiguro et al. Supplementary Figure S2

3. A B C
52.2%
21.5%
15.6%
20 mM DS
Control
2 mM DS
50 mM
DEAB + -
0.2%
ALDH activity
relative cell growth
Control
20 mM DS
2 mM DS
(days)
Control
20 mM DS
2 mM DS
***
*** E F
Control
20 mM DS
ALDH1A1
SOX2
relative RNA levels
z-VAD-FMK
100mM
50mM
20mM Disulfiram
DMSO
active caspase-3
Actin
SOX2
ALDH1A1
Cell viability
(%) D
n.s.
n.s.
ALDH1A1
SOX2
Actin DS
(mM)
Fig. S3. ALDH activity is required for SOX2 expression and proliferation of ovarian CSC-like cells (#2)
Ishiguro et al. Supplementary Figure S3

4. A B C
43.2%
3.8%
6.3%
Control
ALDH1A1-sh2
ALDH1A1-sh3
ALDH activity #4 #2 #5
#20
#5/#4 ratio
#5/#2 ratio
probeID
ALDH1A1
0.178
3.102
26.748
2.571
8.622
A_23_P83098
0.071
0.446
4.108
0.399
57.675
9.209
A_33_P
ALDH1A2
0.007
0.006
0.055
7.854
9.086
A_24_P73577
ALDH1A3
11.441
0.192
0.421
33.293
0.037
2.197
A_23_P205959
ALDH1B1
0.779
0.828
0.553
1.303
0.709
0.668
A_24_P64233
ALDH1L1
0.317
0.022
0.271
3.485
0.069
A_23_P258887
ALDH1L2
0.611
0.042
0.305
0.352
0.499
7.252
A_33_P
ALDH2
7.925
0.377
0.138
0.601
0.017
0.366
A_23_P36753
ALDH3A1
4.258
0.549
0.417
3.079
0.098
0.759
A_33_P
2.747
0.168
0.202
1.778
0.073
1.200
A_23_P207213
ALDH3A2
6.018
3.367
13.369
12.772
2.222
3.971
A_33_P
3.452
3.450
10.229
7.527
2.964
2.965
A_33_P
ALDH3B1
0.226
0.256
0.147
1.130
1.264
A_33_P
0.566
0.355
0.427
0.097
0.753
1.201
A_24_P218688
0.301
0.231
0.247
0.076
0.820
1.065
A_33_P
ALDH3B2
0.072
0.322
0.253
3.518
0.787
A_23_P24311
ALDH4A1
8.443
4.685
4.882
5.541
0.578
1.042
A_23_P170337
0.315
0.265
0.112
0.881
0.354
A_32_P192970
ALDH5A1
0.296
2.240
0.259
13.362
7.568
A_24_P115007
ALDH6A1
3.670
2.296
2.242
1.546
0.976
A_23_P128967
ALDH7A1
11.396
15.791
4.474
17.093
0.393
0.283
A_23_P70231
0.196
1.888
0.173
0.193
0.885
0.092
A_33_P
0.341
1.493
0.333
0.183
0.975
0.223
A_33_P
ALDH8A1
0.051
0.047
0.013
0.254
0.277
A_24_P283324
ALDH9A1
2.981
1.759
3.506
2.067
1.176
1.993
A_24_P385280
ALDH16A1
4.277
3.804
11.184
3.800
2.615
2.940
A_24_P5743
ALDH18A1
1.125
3.981
6.138
1.262
5.458
1.542
A_23_P1361
ALDH1A1-sh2
ALDH1A1-sh3
Control
ALDH1A1
SOX2
Actin D E F G
Control
ALDH1A1-sh3
ALDH1A1-sh2
relative cell growth
(days)
ALDH5A1
ALDH18A1
ALDH1A2
ALDH3A2
ALDHlow
ALDHhigh #5
Actin
ALDH1A1
Control
ALDH1A1-sh2
ALDH1A1-sh3
Control
sh2
sh3
relative RNA levels
SOX2
fig. S4. ALDH1 mediates SOX2 expression and proliferation of ovarian CSCs (#2)
a. 20uM DS ALDH activity
b. 20uM DS spheroid pic
c. 20uM DS cell growth curve
***
***
Ishiguro et al. Supplementary Figure S4

5. A B C D
Control
SOX2-sh5
SOX2-sh1
relative cell growth
(days)
control
Sox2
relative caspase activity
***
Control
SOX2-sh1
SOX2-sh5
SOX2
ALDH1A1
Actin
Control
SOX2-sh1
SOX2-sh5
***
*** E F G H
pSin-control
pSin-Sox2 #4
ALDH1A1
SOX2
Oct-3/4
Nanog
CK7
Actin
ALDH1A1
SOX2
ALDH1A1
CSC
proliferation
Control
Sox2
relative cell growth
(days)
***
fig. S5. ALDH1 mediates SOX2 expression and proliferation of ovarian CSCs (#2)
a. 20uM DS ALDH activity
b. 20uM DS spheroid pic
c. 20uM DS cell growth curve
relative RNA levels *
control
Sox2
Ishiguro et al. Supplementary Figure S5