A B Pax8 CK7 CA125 HE4 HE xenograft tumor primary #20 bright phase HE

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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

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 Y27632 + 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

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 0 2 5 20 (mM) Fig. S3. ALDH activity is required for SOX2 expression and proliferation of ovarian CSC-like cells (#2) 52 33 66 59 68 58 Ishiguro et al. Supplementary Figure S3

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 2.571 150.594 8.622 A_23_P83098 0.071 0.446 4.108 0.399 57.675 9.209 A_33_P3379644 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_P3282634 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_P3238433 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_P3336617 3.452 3.450 10.229 7.527 2.964 2.965 A_33_P3336622 ALDH3B1 0.226 0.256 0.147 1.130 1.264 A_33_P3236563 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_P3367447 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_P3330498 0.341 1.493 0.333 0.183 0.975 0.223 A_33_P3330503 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 0 1 4 7 (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

A B C D Control SOX2-sh5 SOX2-sh1 relative cell growth 0 1 4 7 (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