3 5 - Race Result of Human OTAG-12B from Normal Ovary

Supplementary Figure 1

3’5’- Race result of human OTAG-12b from normal ovary Human OTAG-12b, 1463bp CGAAGCACTGGAGAGTGTCATGGAGGCCTACGAGCAGGTCCAAAAGGGACCCCTGA AGCTGAAAGGCGTCGCAGAGCTGGGAGTGACCAAGCGGAAGAAGAAAAAGAAGGA CAAAGACAAAGCGAAACTCCTGGAAGCAATGGGAACGAGCAAAAAGAACGAGGAG GAGAAGCGGCGCGGCCTGGACAAGCGGACCCCGGCCCAGGCGGCCTTCGAGAAAA TGCAGGAGAAGCGGCAAATGGAAAGGATCCTAAAGAAGGCATCCAAAACCCACAA GCAGAGAGTGGAGGACTTCAACAGACACCTGGACACACTCACGGAGCATTACGACA TTCCCAAAGTCAGCTGGACGAAGTAGCCGCCTGCCCCCAGTATGGAGCAGCATCGA GGGTTCGCAAAAGGCCACACTGGGGTTGTGTGTGTTTCCTTTGGTATATTCTGGAAA CATGGCTACACACACCCTTGCATCTTCTGCTACAGACTGCTTTTCGAAGCTGTGTACC CTCATTCTGGAACTTGATTAAAGTAAGATCGTCCTTGTACTCAGTTTAGGCTTCTTGG CAACATACAGAAGATACACCCTTTTCGTTTGGATGGAAAGTTTCTAAGTTTATCCAG AGGTAAAGCCCATTGTGTGTCTGTGTCATGTAAAAATGTTTTCACCCGAGTTGCATG TAATGCTCTGAGGCCAGCCAGCTGTCTTCTCCTGGATGAGACAGACTCCAGAGGGTA AGGAGCTAGCGCCATGGTGGCCTGCAGTATGCAGAGCCCCGCAGGACCCAGCGTGG GCGCCCTTCCAAGCTTCCTCTAGCTTTGGGCCTATGCTGTCCCCTGCAGGCCCTAGG GAAGCCACTTGCAACTATGCGGCCTTCAGACTTCCTCCTCAGCCACCTGGCCACTGA GACAGCATAGCCTGGGTAACGGAACAGCCACCTAAGGCAAGAATGGAACGGACAC ACCTTGCTCCTTTCTGAGCCCGTTTCCCAAAACCCCCCTTCCAGGTGCTTCTAATGGG TGTTGCCATAGCAGACGCTGCTAATGCATCACAGCATTCTTTGAAATGGAACCAGAC ACAGCCTGCCTCTCAATCCTCAGCTGGGGGCTCCTAGCAGCCTCTTGTATTTACTCA GAGTTGACACATCACACAGATCCTGTTTGGCATTCCTACCTTACGGACGTCTCAGGG GTGACAGGACCAGGGCAGAGCCCCGGTACAAACAGACAAGGCTGCAGTCAAATGG GAGGGTCCAGGTGTCCGTGTTGGAGGGCTGGGATCTTGTAGGGCCTGTGCGTCCTGG CTGAGGATCAAACCACATATGTTATTGGGAGAAACGATTTCTGTTGACGTAGATATT GAAAGAATAATGAAGGCAGAAGAGAAAAACGAAGTGTGGAATTTGGGGTTGTCCT GTGTAAATTACACAATAAAGCAAAAGTCAGTTATTGTAAAAAAAAAAAAAAAA

1 Supplementary Figure 2

Part 1: Amino acid Sequence Comparison of OTAG-12 in different species. Sequences other than mouse and human (our data) are hypothetical as found in data banks. NS- Nuclear localization signal. * indicates conserved positions in all species. NLS

Part II. The underlined sequence shows divergence at the carboxyl-terminal. DNA binding sites are shown in bold.

Amino acid sequence of Human OTAG-12b MEAYEQVQKGPLKLKGVAELGVTKRKKKKKDKDKAKLLEAMGTSKKNEEEKRRGL DKRTPAQAAFEKMQEKRQMERILKKASKTHKQ RVEDFNRHLDTLTEHYDIPKV SWTK

Amino acid sequence of Human OTAG-12a MEAYEQVQKGPLKLKGVAELGVTKRKKKKKDKDKAKLLEAMGTSKKNEEEKRRGLD KRTPAQAAFEKMQEKRQ GHQTSCT A TGFQGHKANAARSY AWTQNWYCVPSS

Supplementary Figure-3 Q-PCR analysis of expression of hOTAG-12a and hOTAG-12c in human ovarian samples (Normal ovaries 11, benign tumor 2 and ovarian cancer 11). hOTAG-

12a expression level in normal ovary is similar to that of human OC samples, while hOTAG-12c expression level in normal ovary is significantly higher than that of human ovarian cancer samples. Normalized by GADPH. * p<0.05.

Supplementary Figure-4 Histology of tumors from xenografted mice. Representative tumor tissue samples shown in Figure 7G were fixed in formalin and paraffin-embedded. Sections were stained with hematoxylin and eosin. Panels a, c and e: without DOX treatment; b, d and f:

DOX treatment; a and b: vector; c and d: hOTAG-12a; e and f: hOTAG-12b-L. Inserts: Bottom left: Tumor mass; Upper right- histology higher magnifications.

Supplementary Figure-5 Potential mechanism of hOTAG-12b induced apoptosis. OTAG-12b induces the expression of GADD45α, CIDEB and BAD, while inhibiting Akt-1 and NAIP.

GADD45 α inhibits cell growth by inducing cell cycle G2 arrest, and could also induces cell apoptosis through MAP3K1/JUK/p38 signal pathway. Up-regulation of CIDEB induces cell apoptosis, while BAD, only the BH3 domain pro-apoptotic protein, binds to Bcl-xL to play a role in apoptosis. Akt-1 important for cell growth and survival by PKB pathway, also could phosphorylate BAD to inactivate its pro-apoptotic effect. Akt1 also inhibits caspase-3 activation.

NAIP (Neuronal apoptosis inhibitor protein) is a member of apoptosis inhibitor proteins (AIP) that could inhibit caspase-3 activity. In summary, OTAG-12b over-expression induces cell G2 arrest by increasing GADD45α expression and induces apoptosis by increasing pro-apoptosis genes CIDEB and BAD, while down-regulating anti-apoptotic genes Akt-1 and NAIP.

5 Supplementary Table 1 Table of Primers used for cloning, mutations and conditions Name Primer Annealing Aim Temperature Mouse Left GGGACACAGGACACAGGTCT 56℃ Q-PCR OTAG-12 Right TTAGACCAGGAATGCCAACC Mouse Left CCACGAGATCTATGGAGGCCTACGAGCAAGTCCA 62℃ ORF, vector: OTAG-12 Right ACTGGAGTCGACCTTGGTCCAGCTGACTTTGGGAAT pIR-V5 Human Left ACGAGCAAAAAGAACGAGGA 56℃ Q-PCR OTAG- Right GCATAAGACCTGGCAGCATT 12a Human Left GTGACCAAGCGGAAGAAGAAA 56℃ Q-PCR OTAG- Right CTCCTGCATTTTCTCGAAGG 12b Human Left CCCCCTTAGACCTTTTCTCG 56℃ Q-PCR OTAG- Right TCCTCGTTCTTTTTGCTCGT 12c human Left ATATTCCTCGAGATGGAGGCCTACGAGCAGGTCCA 62℃ ORF vector: OTAG- A pIR-V5 12a Right ATTTACCGGTACTCGAAGGAACACAGTACCA Human Left ATATTCCTCGAGATGGAGGCCTACGAGCAGGTCCA 62℃ ORF, vector: OTAG- A pIR-V5 12b Right ATTTACCGGTCTTCGTCCAGCTGACTTTGGGAAT hOTAG- Left ATATTCCTCGAGATGGAGGCCTACGAGCAGGTCCA 62℃ ORF, vector: 12b ∆C10 A pIR-V5 Right ATACCGGTATGCTCCGTGAGTGTGTCCA hOTAG- Left ATATTCCTCGAGATGGAGGCCTACGAGCAGGTCCA 62℃ ORF, vector: 12b ∆C21 A pIR-V5 Right ATACCGGTGAAGTCCTCCACTCTCTGCT hOTAG- Left ATATTCCTCGAGATGGAGGCCTACGAGCAGGTCCA 62℃ ORF, vector: 12b ∆C37 A pIR-V5 Right ATACCGGTTTCCATTTGCCGCTTCTCCT Human Left ATTCCTCGAGATGTCTTTTTCCAGGAAGAAGA 62℃ ORF, vector: OTAG- Right ATTTACCGGTACTCGAAGGAACACAGTACCA pIR-V5 12c Human Left AAGAATTCATGGAGGCCTACGAGCAGGT 62℃ ORF, vector : OTAG- Right TATCTAGATCACGTAGAATCGAGACCGA LITMUS 12a and b 29/TO Human 3`RA GCCTGGGTAACGGAACAGCCACCTAA 68℃ RACE OTAG- CE 12b 5`RA GCTGAGGATTGAGAGGCAGGCTGTGT CE

6 Supplementary Table 2 Primers designed for verification of Apoptosis related genes and conditions

Amplico Primer PubMed n Amplicon Primer Sequences Assay Set Reference Boundari Size es Lift: CGAGTGAGCAGGAAGACTCCA ℃ 96 - 443 BAD 12894513 56 348 bps Right: AGGAGTCCACAAACTCGTCACT good nt Lift: TGCTTCAGGGTTTCATCCAG ℃ 146 - 315 BAX 12580957 56 170 bps Right: GGCGGCAATCATCCTCTG good nt Lift: ATGTGTGTGGAGAGCGTCAACC ℃ 500 - 695 Bcl2 11727956 Right: 60 196 bps nt TGAGCAGAGTCTTCAGAGACAGCC good Lift: GGGGTAAACTGGGGTCGCATT 766 - Bcl-xl 11727956 no 237 bps Right: ACCTGCGGTTGAAGCGTTC 1002 nt Lift: AGAACTGGACTGTGGCATTGAG ℃ 566 - 756 Casp3 11727956 56 191 bps Right: GCTTGTCGGCATACTGTTTCAG good nt Lift: AGTGACAGGTATGGGCGTTCG ℃ 626 - 899 Casp7 11727956 60 274 bps Right: GCATCTATCCCCCCTAAAGTGG Good nt Lift: CTTGATGGAGACCCTCCTGTATG ℃ 93 - 183 BIK 11756183 56 91 bps Right: AGGGTCCAGGTCCTCTTCAGA good nt Lift: TCAGTAACTGGGAACCAAAGGATG ℃ 2079 - BIRC2 12894513 Right: 60 401 bps 2479 nt CAACAGCTGTTCAAGAAGATGAGG good Lift: ACTACATAGGACCTGGAGACAGAG ℃ 806 - BIRC3 12894513 Right: 56 382 bps 1187 nt AAGTACTCACACCTTGGAAACCAC good Lift: GGCAGACCAGCATGACAGATT ℃ 663 - 735 P21 11750059 56 73 bps Right: GCGGATTAGGGCTTCCTCTT Ok nt Lift: TCAGCGCACGATCACTGTC ℃ 391 - 473 GADD45α 11750059 56 83 bps Right: CCAGCAGGCACAACACCAC ok nt Lift: CATGGAGACGAGGACACGTACTAC ℃ 1155 - Tp73 NM_005427.2 56 93 bps Right: CTCCATCAGCTCCAGGCTCT ok 1247 nt Lift: TGTGTCAGCCCTGGACTACC ℃ 969 - Akt1 12894513 56 383 bps Right: TGAGCAGCCCTGAAAGCAAG good 1351 nt NOD1 Lift: CCAGAATCAGATCACAGCTAAGG ℃ 3235— NM_006092.2| 56 176 bps Right: ACCCCATGAACAGGAAAGC good 3410 nt ℃ Lift: TGCTGGATGACAAGGAAGAG 56 883-- CIDEA NM_198289.2| 120 bps Right: GAGCCAAACGAAACACAGTG Not 1002 nt good Lift: AATTTCCTCCACCCTTCGTC ℃ 1721-- CIDEB NM_014430.2| 56 178 bps Right: GTTATCTAGCCTGTACTGTCTGC good 1898 nt Lift: GGTGACCTGCAGAATCCTAAG ℃ 2405-- DFFA NM_213566.1| 56 191 bps Right: CGTATGTTGAGACCTGGAATAGC good 2595 nt NM_001126117 Lift: TCATGTTCAAGACAGAAGGGC ℃ 1267-- P53 56 178 bps .1| Right: CGGGACAAAGCAAATGGAAG good 1090 nt Lift: ATGTTTCAGACGGTTCCCC ℃ 990-- P27 NM_004064.3| 56 194 bps Right: CATTCCATGAAGTCAGCGATATG good 1183 nt TRAF2 NM_021138.3| Lift: GCAAAGAATTCCTACGTGCG 56℃ 1495-- 164 bps

7 Right: CACTTGTACAGTGAGACCCTG good 1658 nt Lift: CTAGGCTGGTCTTGAACTCTTG ℃ 56 2754--- APAF1 NM_181869.1| Right: GCAGCTACAGTGAGAAAAGGG 175 bps Not 2928 nt good Lift: CGACTACCAAGGCTCATTAGAC ℃ 4101-- NAIP NM_022892.1| Right:CCAAGACAAATTATTGATTCAGCA 56 201 bps 4301 nt G good Lift: ACGACATCTTTGAGCAGTGG ℃ 748--927 CASP9 NM_032996.1| 56 180 bps Right: CAAGATAAGGCAGGGTGAGG good nt NM_001080125 Lift: AAACAGATGCCTCAGCCTAC ℃ 1759-- CASP8 56 191 bps .1| Right: GAGAATGGCCTGAACCCG good 1949 nt