SUPPLEMENTAL INFORMATION

SUPPLEMENTARY MATHERIALS AND METHODS

Plasmid construction.

The human CREB1 was amplified using F:AGTCGAATTCAATGACCATGG and

R:AGTCCTGCAGTTTCCTCATTT primers, then cloned in frame to EGPF into pME-EGFP (383,

Tol2kit)1 between EcoRI and PstI restriction sites. The p5’-228-pu.1 promoter plasmid was

obtained by excising the 9-kb zebrafish pu.1 promoter sequence from 9-zpu.1-GFP plasmid 2 using

SalI and SacII restriction enzyme (NEB, Ipswich, MA), and by cloning it into p5E MCS (228) 1.

The plasmids were recombinated through LR reaction (Gateway Technology, Invitrogen-Life

Technologies, Monza, Italy) according to manufacturer’s recommendation, using LR plus clonase

enzyme and performing the reaction for 16 h at 25°C. After LR reactions, 2 µl of the sample was

transformed into TOP 10 bacteria (Invitrogen-Life Technologies). Clones were selected on

carbenicillin plates and screened by PCR, miniprep and DNA sequencing. Entry and destination

vectors used from Tol2kit were: 299 (p5E-bactin2), 228 (p5E-MCS), 383 (pME-EGFP), 302 (p3E-

polyA), 394 (pDestTol2pA2)1. Trasposase mRNA synthesis was performed using mMESSAGE

mMACHINE T7/SP6 kit (Ambion, Austin, TX), using pCS-TP plasmid, kindly provided by Dr.

Koichi Kawasakami. Plasmids used for RNA probes synthesis were obtained by PCR fragments

generated from a pool of cDNAs from zebrafish embryos at different stages (at 24 and at 48 hpf)

and cloned into pCRII-TOPO (Invitrogen-Life Technologies). Probes for and jun were designed

from the published NCBI sequence (NM_199987.1) using F:CACCGCTCTCTCCTATCGAC and

R:TGAGGTGCTCAGAAAACAAACA primers. Bcl2 plasmid used to synthesize riboprobes were

kindly provided from Dr. Forrester AM3.

Whole-mount in situ hybridization (WISH).

1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

12

Digoxogenin (DIG)-labelled antisense zebrafish RNA probes were synthesized according to

manufacturers’ instruction. Briefly, plasmids were linearized with NotI or BamHI restriction

enzyme (NEB) and all antisense riboprobes were synthesized using DIG RNA Labeling Kit

(SP6/T7) (Roche Biochemicals (Indianapolis, IN) Roche, luogo). Whole-mount in situ

hybridization (WISH) for mRNA expression was performed as previously published 4. Briefly,

embryos were fixed in 4% paraformaldehyde (PFA) and the DIG probes were detected using anti-

DIG-alkaline phosphatase (AP) and NBT/BCIP. After in situ hybridization, embryos were put in

4% paraformaldehyde in PBS, and mounted in 85% glycerol/PBS. Embryos were visualized and

imaged with Nikon SMZ1500 zoom stereomicroscope (Nikon Instruments Inc).

Luciferase assay.

For luciferase activity detection, 20 ng/µl of pDest-βactin:EGFP_CREB-polyA or pDest-

βactin:EGFP-polyA together with 20 ng/µl of pAd C6-6xCRE_luciferase plasmids were injected

into the yolk of fertilized one-cell stage embryos. Renilla plasmid was used at 1:10 ratio. 26 h after

injection, proteins were extracted and analyzed by Dual Luciferase Assay System (Promega

Corporation, Madison, WI).

AML score.

We scored the AML on 77 CREB-zebrafish by using different methods, and when it was possible,

also by confirming diagnosis with two methods for the same fish (such as MGG+RNA or

MGG+FACS). In particular, we extracted the kidney marrow and/or mass for 16 fish and MGG and

ANAE staining was evaluated; in 8 fish we dissociated kidney marrow and/or mass and cells were

analyzed by FACS; 25 whole fish were paraffin-included and sagitally sectioned for histological

evaluation by H&E, CREB, PCNA, l-plastin staining; from 5 kidney marrow we extracted RNA

and perform gene expression profiling; 27 fish were found dead with abdominal mass.

2

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

12

Hisytology, immunohistochemistry and cytology.

Whole adult zebrafish were fixed in 10% neutral buffered formalin, embedded in paraffin, and 5 μm

serial sections were obtained. Hematoxylin and eosin (H&E) staining, and immunohistochemical

analyses were performed. For this purpose, sections were re-hydrated and then antigen retrieval was

performed by incubation with citrate buffer 0.01 M pH6 at 95 °C for 20 min. For

immunohistochemistry, slides were incubated with 1:500 dilution anti-CREB (AB-129 Sigma-

Aldrich), 1:500 dilution anti-PCNA (FL-261, Santa Cruz Biotechnology), 1:500 dilution

anti-C/EBP δ (C-22, Santa Cruz Biotechnology) for 1 hour and then developed with the DAKO

Envision Detection Kit (Dako, Carpinteria, CA). Kidney or abdominal mass isolated from zebrafish

were imprinted on glass, and stained with MGG and Alpha-naphtyl acetate esterase (ANAE) with

standard procedures. Images were captured on a Nikon SMZ1500 zoom stereomicroscope (Nikon

Instruments Inc), or on a ViCo Nikon eclipse 80i microscope.

Reverse phase protein arrays and Western Blot.

RPPA analysis was performed as described previously5. In brief, a previously analysed cohort of 66

AML patients6 samples were stained for total protein content (Fast Green FCF; Sigma-Aldrich) and

with 5 different previously validated antibodies using the Catalyzed Signal Amplification System

kit (Dako). The images of stained slides were analyzed using the Microvigene software

(VigeneTech Inc, Boston, MA) to generate normalized and background-subtracted single values for

each protein in each sample. CREB S133 (Cell Signaling) and C/EBPδ (C-22, Santa Cruz

Biotechnology), were previously subjected to extensive validation for single band specificity by

Western blot analysis. For Western Blot experiments, total proteins lysates were obtained by

resuspending cell pellet in ice cold lysis buffer (Biosource International; Camarillo, CA)

supplemented with 0.5 mM PhenylMethaneSulfonyl Fluoride (PMSF, Sigma-Aldrich), mammalian

protease, and phosphatase inhibitor mixture (both 1×, Sigma-Aldrich), for 30 min on ice. Total

3

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

12

proteins lysates (15 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel

electrophoresis. Immunoblots were hybridized with anti-CREB (AB-129), anti-β-actin (AC-74,

both Sigma-Aldrich), anti-CREB S133 (Cell Signaling) and anti-C/EBPδ (C-22, Santa Cruz

Biotechnology). Enhanced chemiluminescence western blot detection reagents and films (Bio-Rad,

Hercules, CA) were used. Densitometric analyses for protein quantification were carried out using

the UVIBAND Image quantification software (Uvitec, Cambridge, UK). The value of each band

was normalized to the value of β-actin protein.

Cell lines, primary cell cultures and transfection.

Cell lines were purchased by DSMZ. HBM and AML primary cell cultures were obtained from BM

from healthy donors or from AML patients respectively. In compliance with the Helsinki

Declaration, informed consent was obtained from the parents of the patients. The cells were

cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen)

and the following recombinant human (rh) cytokines: interleukin-3 (rhIL-3; 20 ng/mL), interleukin-

6 (rhIL-6; 20ng/mL), stem cell factor (rhSCF; 50 ng/mL), thrombopoietin (rhTPO; 50 ng/mL) and

fms-like tyrosine kinase-3 ligand (rhFLT-3 ligand; 50 ng/mL). All cytokine were from Miltenyi

(Miltenyi Biotec, B. Gladbach, Germany) Cell transfection was performed using a Amaxa

Nucleofector Biosystems (Lonza; Cologne, Germany) according to the manufacturer's guidelines

and efficiency was up to 40%. Healthy bone marrow cells were transfected with 2 µg of pEGFP-

N1- ΔGFP -CREB plasmid or an empty vector (pEGFPN1-ΔGFP-EV) used as control. AML

primary cells and HL60 were silenced for CREB by a mix of 2 exogenous small interfering RNAs

(siRNA) specific for the CREB gene (siRNA1+siRNA3 (37), 100 nmol/L, Dharmacon Industries,

Lafayette, CO). HL60 were silenced for C/EBPδ gene by IBONI siRNA pool (100 nmol/L,

Euroclone). A siRneg was used as control. C/EBPδ was overexpress in HL60 by transfecting

DNA3HA-CEBPD plasmid kindly provided by Dr. Ju-Ming Wang8.

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

12

RNA extraction and SYBR Green quantitative real-time reverse transcription-PCR assays.

Total RNA was extracted from cells using Trizol (Invitrogen-Life Technologies). RNA (1 μg) was

transcribed using the SuperScript II system (Invitrogen-Life Technologies) following the

manufacturer's instructions. RQ-PCR was performed with 1 μL cDNA in 20 μL using the SYBR

Green method (Invitrogen-Life Technologies) and analyzed on an ABI PRISM 7900HT Sequence

Detection System (Applied Biosystems-Life Technologies). Expression values were normalized to

GUS and expression as fold change relative to the control was calculated by the comparative ΔΔCt

method9.

5

1

2

3

4

5

6

7

8

9

10

11

12

12

SUPPLEMENTARY TABLES AND FIGURES

Supplemental Table 1: 258 differentially expressed genes between CREB-zebrafish and controls

with p-valueBH (Benjamini Hochberg method) <0.05.

ProbeSymbol Means for Group CREB-zebrafish Means for Group

ControlDr.26132.1.S1_at pck1 9,630376109 3,828598094Dr.460.1.A1_at pvalb2 10,66077218 4,900338762DrAffx.1.80.S1_at pvalb4 9,587280877 4,516676484Dr.15359.1.S1_at pvalb3 11,4650376 5,516140631Dr.20928.1.S1_at pvalb1 10,46721311 5,036368241Dr.4812.1.S1_s_at 9,455019809 4,578641744Dr.8472.1.S1_at tnnc2 8,885524679 4,529379082Dr.20815.1.S1_at tpma 8,364042465 4,38057195Dr.10620.1.S1_at tnnt3b 10,16647744 5,376442217Dr.11351.1.A1_at zgc:73324 7,790952155 4,16401722Dr.11552.1.S1_at atp2a1l 9,824941034 5,285935187Dr.18267.1.S1_at ckma 10,69869148 5,778254885Dr.24260.1.S1_at mylz3 10,06059281 5,448457447Dr.12986.1.A1_at fos 9,06976537 5,185475263AFFX-Dr-acta1-3_at actc1b 11,21856938 6,425097431Dr.24950.1.S1_at ckmb 11,62885856 6,662155658Dr.2022.1.A1_at arg2 10,03737884 5,754437887Dr.676.1.S1_at slc25a4 8,173065435 4,72309271Dr.1967.1.A1_at wu:fb51h06 7,906490587 4,612606495Dr.2675.1.A1_at fkbp5 10,61225801 6,216611446DrAffx.1.68.S1_at tnni2a.3 8,213870742 4,829114297Dr.2914.1.S1_at mylz2 9,404060929 5,563622806Dr.26319.1.A1_at ddc 10,94377991 6,500497766Dr.24941.1.S1_at 10,81275331 6,447232737Dr.2914.1.S2_at mylz2 8,203554967 4,918869228Dr.8143.1.S1_at fabp2 8,088336784 4,866382574Dr.5066.1.S1_at actn3b 7,419614349 4,465626798Dr.18853.1.A1_at wu:fc22g01 8,834068744 5,323093072Dr.1448.1.S1_at myl1 6,366472922 3,852610115Dr.4095.1.A1_at ass1 9,833037784 5,951366392Dr.5687.1.A1_at 8,919772859 5,438561548Dr.1662.1.S1_at ttnb 7,065181401 4,316260728Dr.815.1.S1_x_at actc1b 11,08773295 6,878448681Dr.861.1.S1_at ddc 5,685444498 3,53883369Dr.815.1.S1_at actc1b 11,16313551 6,95761899Dr.11483.1.S1_at ldb3b 6,73179121 4,218290659Dr.4681.1.A1_at ttna 6,642935403 4,195576352Dr.5224.1.A1_at neb 5,685167722 3,605603145Dr.23911.1.A1_at slc20a1a 11,40775168 7,242559479

6

123

4

5

12

Dr.26339.1.A1_at 8,619267365 5,477235411Dr.12986.1.A1_a_at

fos 10,18747622 6,492783929

Dr.7140.1.A1_at cx28.9 7,151270521 4,564975992Dr.18442.1.A1_at zgc:152984 7,071635725 4,519918102Dr.20990.2.S1_at ttna 7,604660752 4,863482045Dr.22425.1.A1_at wu:fj67g12 5,981440829 3,857486073Dr.7502.1.A1_at zgc:165344 8,016001472 5,169944896Dr.9243.1.A1_at nr4a1 8,622529168 5,56758083Dr.13817.1.A1_at 5,982339814 3,893870295Dr.13267.1.A1_at LOC100330152 4,456465568 2,903359041Dr.15596.1.A1_at zgc:194246 5,766378349 3,79586605Dr.16470.1.A1_at 6,311404594 4,179219914Dr.21800.1.A1_at mybpc2b 6,722590813 4,48890829Dr.22685.1.A1_at dusp2 9,162721962 6,127411414Dr.16051.1.S1_at pdlim7 6,645734279 4,450264145Dr.9294.1.A1_at wu:fc14c01 4,960662631 3,327233358Dr.1580.1.A1_at aldoab 8,273320452 5,648905122Dr.1659.1.A1_at 9,629317576 6,59694005Dr.22971.1.A1_at si:ch211-

235e18.39,824257954 6,742031891

Dr.18614.1.A1_at 7,781739345 5,346049315Dr.1816.1.A1_at zgc:92806 6,548179447 4,501824766Dr.9996.1.A1_at emx2 7,80092349 5,405941736Dr.15501.1.S1_at zgc:85866 9,503800028 6,596727927Dr.19499.1.A1_at 7,896375272 5,499957382Dr.19512.1.A1_at 5,586444111 3,893319222Dr.20198.2.S1_x_at hsp70 8,402403051 5,872059403Dr.7666.2.A1_at zgc:158791 6,395252949 4,475412001Dr.4097.1.S1_at zgc:63514 7,956234502 5,570924893Dr.13833.1.S1_at 7,629484485 5,356774825Dr.17476.1.A1_at 5,239655996 3,698986814Dr.6286.1.A1_at 6,188848809 4,385341357Dr.26516.1.S1_at 7,743686861 5,508270657Dr.18643.1.A1_at 11,43146077 8,135412346Dr.24351.1.A1_at wu:fb08c08 6,320163488 4,500000783Dr.17619.1.A1_at zgc:112054 6,280527019 4,479310903Dr.17579.1.A1_at 8,91795703 6,381224723Dr.13165.1.A1_at npr3 5,109707284 3,658978129Dr.25427.1.A1_at 7,259824013 5,203951153Dr.19928.1.A1_at ppargc1b 9,13661269 6,561544682Dr.16174.1.A1_at 8,989154232 6,456057035Dr.13001.1.A1_at esrp1 7,405662143 5,337720238Dr.5745.1.A1_at 5,995724395 4,321566208Dr.5663.1.S1_at 8,752508806 6,311625407Dr.11177.1.A1_at 8,464688467 6,104647927Dr.25095.1.A1_at thbs2b 4,902721216 3,536790643

712

Dr.8798.1.A1_at wu:fc83d12 5,935492539 4,282206147Dr.18919.1.A1_at 5,876481475 4,24350191Dr.25929.1.A1_at 9,297250403 6,724037284Dr.25769.1.S1_at hsd17b3 4,504586664 3,264403956Dr.23766.1.A1_at wu:fa55d03 5,568687826 4,043364932Dr.7608.1.A1_at jun 9,577947421 6,967543476Dr.2625.1.A1_at dst 9,216735881 6,707529731Dr.22059.2.A1_at 8,192356456 5,962321149Dr.25767.1.S1_x_at 11,70309892 8,523573048Dr.37.1.S1_a_at pou3f3a 5,670594122 4,133441377Dr.9896.1.A1_at si:ch211-

195b13.17,703495657 5,623993576

Dr.4864.1.A1_at sccpdha 5,84845414 4,271801113Dr.26281.1.A1_at rgl3a 8,766488537 6,420482357Dr.23813.1.S1_at zgc:110409 5,552340237 4,067539618Dr.14365.1.S1_at 8,10670477 5,943145738Dr.7714.1.S1_at etv5b 8,744817213 6,411108865Dr.6321.1.A1_at fasn 8,401780539 6,184917531Dr.22170.1.A1_at slc6a19a 8,039877279 5,91937545Dr.796.1.S1_at 7,840945601 5,776875948Dr.7142.1.A1_at 7,417352061 5,465129722Dr.13756.1.S1_at 10,69941546 7,887737559Dr.4271.1.S1_at slc13a2 5,723717992 4,223711625Dr.26491.1.S1_at slc43a2a 7,821451008 5,774959101Dr.9217.1.A1_at 6,056438387 4,472110418Dr.25257.1.A1_at bhlhe41 9,76285343 7,22271672Dr.26432.1.A1_at 8,94684626 6,622409507Dr.25767.1.S1_at 12,101918 9,010603824Dr.13007.1.A1_at zplxdc2 5,685008017 4,232830971Dr.18271.1.S1_at 7,860491223 5,866738738Dr.21068.1.S1_s_at pou3f3b 6,154648518 4,594026286Dr.26261.1.A1_s_at

8,779942394 6,55393033

Dr.22420.1.A1_at hnf4g 8,590322537 6,416521705Dr.19816.1.S1_at LOC792958 7,263261614 5,427125067Dr.6754.1.A1_at per2 9,872484723 7,387438988Dr.26517.1.S1_at pgam2 6,361188632 4,769569449Dr.20028.1.A1_at pgpep1 5,821527354 4,367930545Dr.26095.1.A1_at 10,52029666 7,894139616Dr.20197.1.A1_at slc43a2a 11,33620551 8,520537873Dr.2490.1.A1_at wu:fj66a01 9,445400502 7,1028476Dr.1280.1.A1_at cebpd 10,08314919 7,608931548DrAffx.1.22.S1_at rhcgb 6,861983347 5,186143406Dr.24461.1.A1_a_at

faah 8,743962881 6,610976615

Dr.1137.1.A1_at ier2 9,804379228 7,419771377Dr.3563.1.A1_at tm4sf4 8,463350873 6,410325863

812

Dr.13121.2.A1_at zgc:91897 7,608097313 5,766285465Dr.16231.1.A1_at 6,530691494 4,953051207Dr.737.1.A1_at junbl 9,874285424 7,493114773Dr.26292.1.A1_at wu:fj68b04 9,589006154 7,27883409Dr.12493.1.S1_a_at si:ch211-57h10.1 6,271452895 4,760730839Dr.4011.1.S1_at sb:cb11 7,744633347 5,885092242Dr.15255.1.A1_at sik2b 9,113586847 6,926025902Dr.12489.1.S1_at LOC553527 5,848989323 4,452661356Dr.26174.1.A1_at 7,537359211 5,755960472Dr.16307.1.A1_at 9,018335012 6,904274474Dr.14132.1.S1_at 6,470428548 4,95575086Dr.12975.1.A1_at 9,946395163 7,62326713Dr.19556.2.S1_at 8,363654065 6,415461342Dr.11110.1.A1_at zgc:162707 6,176598657 4,740513677Dr.916.1.S1_at aqp8a.1 7,622855714 5,853040635Dr.23338.1.A1_at 7,177564818 5,515533054Dr.10128.1.A1_at wu:fl02d04 7,79842193 5,99929625Dr.18817.1.A1_at 7,462201689 5,742193756Dr.13121.2.A1_x_at

zgc:91897 6,913296501 5,321182515

Dr.13050.1.S1_at phlda2 10,38999997 7,997473912Dr.610.1.S1_at hsp90a.1 5,886378375 4,534450311Dr.14438.1.A1_at usp2a 6,977992301 5,377160366Dr.10568.1.A1_at 9,552476411 7,365840964Dr.18239.2.A1_at 7,920007088 6,107670851Dr.17070.1.A1_at 8,399140194 6,486815892Dr.13178.1.A1_at bcar1 6,94642296 5,37006363Dr.9986.1.A1_at grtp1a 7,911218267 6,116560582Dr.11252.1.A1_at zgc:56085 6,368727256 4,925634887Dr.13890.1.A1_at 8,179894533 6,33721344Dr.25536.1.A1_at hsp90a.2 9,73587874 7,543490986Dr.10032.1.S1_at zgc:92851 7,755904177 6,015633065Dr.26154.1.A1_at 6,403640144 4,967926259Dr.24499.1.A1_at 7,503183408 5,829763113Dr.5000.1.A1_at si:ch73-46j18.5 8,598136097 6,696215789Dr.18069.1.S1_at LOC100332904 7,017600898 5,467213248Dr.22174.1.S1_at wu:fd60h05 7,440142177 5,801603179Dr.22306.1.S1_at 5,999614279 4,678744894Dr.12819.1.A1_at 7,727694341 6,034335044Dr.18722.1.A1_at 7,419633796 5,804458783Dr.11374.1.A1_at ahcyl2 8,461839753 6,623595359Dr.20856.1.S1_at 7,898235695 6,183713568Dr.22172.1.A1_at 7,828048519 6,129261462Dr.17610.1.A1_at 7,960074907 6,235393605Dr.12531.1.S1_at coq10b 8,74537139 6,852660135Dr.1378.1.S1_at gadd45ba 10,81897977 8,487293413Dr.6517.2.S1_at 9,048241317 7,10066002

912

Dr.12953.1.A1_at 7,945201218 6,235536245Dr.11445.1.A1_at 8,416858854 6,607092952Dr.2991.1.A1_at trpm7 10,32448959 8,111344206Dr.12802.1.A1_at 7,416634457 5,830564708Dr.16222.1.A1_at 7,758348355 6,102101296Dr.4736.1.A1_at 6,863807821 5,405323186Dr.796.1.S1_x_at 7,511863798 5,917402279Dr.13098.1.S1_at 7,813302355 6,155525311Dr.22479.1.A1_at 7,187202693 5,66250446Dr.25269.1.A1_at wu:fb09c09 7,22147683 5,690081657Dr.22169.1.A1_at zgc:113516 8,072769568 6,366886285Dr.19816.2.S1_at LOC792958 7,880650276 6,218192985Dr.7806.1.A1_at 8,358971851 6,606533161Dr.15101.1.S1_at si:dkey-286j15.1 5,778848105 4,572656826Dr.7710.1.A1_at ets2 7,13170411 5,646978789Dr.15958.1.S1_at 7,541315269 5,972806239Dr.4291.1.A1_at si:ch211-

285f17.17,639004259 6,051509575

Dr.646.1.S1_at mfn2 9,479862218 7,515681135Dr.16022.1.A1_at nit1 8,068012995 6,400554717Dr.5583.1.A1_at 10,95963729 8,705996145Dr.7609.1.A1_at prlra 8,833969064 7,025152885Dr.278.1.S1_at emx2 6,386513874 5,084629498Dr.12995.1.A1_at 10,1623554 8,111155142Dr.12256.1.A1_at slc2a11l 8,59946594 6,868809262Dr.12613.1.S1_a_at irx1a 8,897058369 7,106825538Dr.25228.2.S1_at 7,861856948 6,280813891Dr.26114.1.A1_at 10,58945876 8,468270938Dr.14286.1.A1_at wu:fc03g10 8,445339384 6,753960843Dr.13121.1.A1_at 8,58122327 6,870524338Dr.23764.1.A1_at glis3 8,34352468 6,695924327Dr.10326.1.S1_at junb 10,14747086 8,164097745Dr.18724.1.A1_at cxxc5 7,032828828 5,66194169Dr.4310.1.S1_at 8,196277584 6,603575119Dr.14489.1.A1_at abcc4 8,761546181 7,061763387Dr.12153.1.A1_at rsph9 7,097641062 5,735431375Dr.26169.1.A1_at 7,465341766 6,035534561Dr.2413.1.S1_at dusp1 11,55190641 9,377264983Dr.5779.1.S1_at hoxb3a 8,000430705 6,495799367Dr.4485.3.A1_x_at 10,71495585 8,739378447Dr.1368.4.A1_at 12,41643916 10,13494995Dr.10168.1.S1_at maf 8,419063029 6,873277233Dr.20954.1.S1_at klc1 8,906102694 7,275543644Dr.13121.1.A1_x_at

8,434768812 6,892760439

Dr.24995.4.S1_at gldc 10,29679034 8,424229146Dr.5342.1.A1_at hspb8 9,230054187 7,554561195

1012

Dr.6759.1.A1_at wu:fb81c07 9,052356258 7,416725358Dr.5873.1.A1_at ahcyl1 12,02298327 9,870696286Dr.20705.1.A1_at ap1m2 7,049684978 5,800382374Dr.2710.2.A1_at tom1 8,61944791 7,095018Dr.17145.1.S1_at kctd12.1 8,617053152 7,094681839Dr.7110.1.S1_at 9,408113982 7,754368234Dr.14326.1.A1_at 9,235132911 7,623937979Dr.19364.1.S1_at wu:fb96d05 7,848242736 6,47947065Dr.3205.1.S1_at 8,018537654 6,626584843Dr.4485.3.A1_at 10,54943576 8,721920736Dr.3197.3.A1_x_at chrna2b 8,956901025 7,412946184Dr.2783.1.A1_at pikfyve 7,221177634 5,978979797Dr.16401.1.A1_at slco1f1 7,960497763 6,596011355Dr.14282.1.S1_at atf3 8,295911191 6,881331274Dr.24337.1.A1_at zgc:66337 10,15869072 8,443057629Dr.12961.2.S1_at irf2bp2b 9,405742064 7,839974933Dr.7178.1.A1_at hmg20a 7,88077349 6,569667309Dr.24954.1.A1_at 8,258144811 6,895189409Dr.3614.1.S1_at abcd3a 10,61505125 8,871466432Dr.5578.2.S1_a_at odc1 9,833293542 8,23531038Dr.17497.1.A1_at 11,17791003 9,376723259Dr.12134.3.S1_x_at zgc:162318 9,09680469 7,668986122Dr.1658.1.A1_at slc4a2a 10,82349549 9,136743341Dr.2850.1.S1_at mat2a 11,13678712 9,43616859Dr.1699.1.A1_at pklr 8,553056508 7,251689142Dr.10431.1.S1_at ctgf 10,40031039 8,821567435Dr.885.1.S1_at sb:cb122 10,31951192 8,763144566Dr.15698.1.A1_at si:ch211-

45m15.110,92442089 9,288290841

Dr.16433.1.A1_at mfi2 9,425953304 8,036845606Dr.10624.1.S1_at zgc:110343 10,4249333 8,923581896Dr.10320.1.S1_at sgk1 11,66610006 9,989947633Dr.3936.1.A1_at zgc:162730 11,52848361 9,907717502Dr.23357.1.A1_at si:dkey-33i11.9 11,39848444 9,889386029Dr.2912.1.A1_at 11,14103997 9,67410578Dr.20052.1.S1_at ppib 9,490343244 10,59079362Dr.26428.1.A1_at nucks1a 8,956065459 10,09353069Dr.10477.1.S1_at nsa2 9,75003597 11,19637147Dr.17470.1.S1_at 11,51781831 13,23014204Dr.14477.1.S1_at ssr4 9,693118684 11,2042183Dr.20019.1.S1_at thy1 10,38947679 12,07191456Dr.8647.1.A1_at arfip1 7,663347345 8,910943958Dr.10315.1.S1_at LOC798703 7,659547638 8,98601449Dr.14671.1.S1_at birc5a 8,024193129 9,437958399Dr.24379.1.S1_at cdk1 7,777999403 9,149476681Dr.11241.1.S1_at calm1b 8,132577693 9,567197404Dr.9071.2.S1_at 6,654230582 7,845527113

1112

Dr.3190.1.S1_at zmat2 7,011096911 8,281109803Dr.21260.1.A1_at 9,500359852 11,2242072Dr.11066.2.A1_x_at

zgc:110249 7,388051267 8,729471046

Dr.6424.1.A1_at zgc:194167 7,80139567 9,232736824Dr.16802.1.S1_at vamp8 7,997638168 9,517793879Dr.6099.1.S1_at scinlb 8,938671962 10,67738152Dr.15617.1.S1_at mettl5 6,933042045 8,369623365Dr.20137.1.S1_at ngdn 6,747991011 8,15268242Dr.18696.1.S1_at c20orf11 5,639017983 6,81692293Dr.25187.3.S1_at si:dkey-42i9.4 8,174186167 9,886441773Dr.13731.1.S1_at zgc:86811 6,292303398 7,634729586Dr.21039.1.S1_at rps6ka3a 7,516764059 9,151526361Dr.3337.1.S1_at esco2 6,325781645 7,704153229Dr.7610.1.A1_at alox5ap 8,627107661 10,51241536Dr.11066.2.A1_a_at

6,743741766 8,22997354

Dr.4066.1.S1_at prkcd 8,110893732 9,900125395Dr.6812.1.A1_at zgc:66472 5,337902592 6,52389447Dr.15988.1.S1_at zgc:158852 9,766283383 11,96620507Dr.7805.1.S1_at rogdi 6,893517568 8,46172676Dr.3817.1.S1_at pdia4 8,185274781 10,05052786Dr.17471.1.S1_at 5,369205448 6,597097482Dr.25495.1.S1_at cpsf5 6,533161117 8,044151102Dr.17389.1.A1_at 6,320104586 7,786104574Dr.977.2.A1_at mus81 5,940289813 7,32559385Dr.16595.1.S1_at si:dkeyp-55f12.4 6,551390647 8,086334963Dr.7189.1.S1_at atl3 5,779143606 7,13442711Dr.15033.1.S1_at 8,11260447 10,03049971Dr.3582.1.A1_at zgc:174906 6,780422858 8,495957551Dr.14775.2.A1_at fam122b 5,826561754 7,302619692Dr.9013.1.A1_at zgc:77713 6,519055345 8,177607242Dr.16546.1.S1_at ldlrap1a 6,779924302 8,505659308Dr.6822.1.A1_at lman1 6,069765957 7,62639146Dr.6716.1.S1_at wu:fb97g01 7,698197432 9,676351272Dr.6360.1.A1_at si:ch211-266i6.3 6,524125297 8,21058031Dr.2999.1.S1_at srgn 9,225468981 11,66258828Dr.7155.2.S1_a_at nusap1 6,147448988 7,771692888Dr.2124.1.A1_at si:dkeyp-84f11.5 5,533808447 6,996599Dr.15687.3.S1_at decr1 6,036777714 7,636954087Dr.2039.1.A1_at zgc:165515 5,568885579 7,055287076Dr.17698.1.S1_s_at zgc:92594 5,230438883 6,637031044Dr.25209.1.S1_s_at zgc:64098 6,630949182 8,450479832Dr.14203.1.S1_at ppp1r14aa 6,698645978 8,542325294Dr.11429.1.A1_at zgc:175133 4,861648465 6,217546669Dr.15927.2.S1_at tbpl1 5,612045907 7,182107653Dr.23479.2.S1_at 6,012827397 7,699864005

1212

Dr.11460.1.A1_at rnf180 5,844360166 7,49013343Dr.15888.1.A1_at zgc:91862 5,626149687 7,235222195Dr.14004.1.S1_at cdc34a 6,111942966 7,86089394Dr.22985.1.A1_at timp2b 7,516684056 9,711136896Dr.15005.1.S1_at LOC100150296 5,251231129 6,800927407Dr.15820.1.S1_at LOC100332626 6,892543094 8,93613701Dr.13990.1.A1_at slc25a1 5,852073113 7,599128097Dr.6570.1.S1_at cited3 6,836225168 8,881450343Dr.407.1.A1_at wu:fa11b04 5,344382251 6,952525101Dr.17743.1.A1_at pik3ip1 5,907334819 7,714974845Dr.12252.1.A1_at selp 5,068829449 6,624596821Dr.15751.1.A1_at LOC100148412 5,113298137 6,688659133Dr.4369.1.A1_at wu:fc06d01 5,792575583 7,602304299Dr.25520.1.A1_at fbxo25 5,307157843 6,97758132Dr.3957.1.S1_at pop5 5,274293436 6,935172375Dr.17557.1.S1_at ncalda 6,047178635 7,956475195Dr.4128.1.A1_at wu:fb34c08 5,221916909 6,897791529Dr.25407.1.A1_at cuedc2 5,268162953 6,974097169Dr.15641.1.A1_at csnk1g2b 5,059257335 6,738125499Dr.23936.1.A1_at si:rp71-1g18.1 5,068974808 6,760038439Dr.23479.1.S1_a_at 5,547566472 7,400811693Dr.24938.1.S1_a_at zgc:158463 8,288712201 11,06776093Dr.7467.1.S1_at serinc5 5,779524576 7,720332453Dr.25788.1.S1_at il13ra2 5,069191101 6,804738463Dr.17935.1.S1_at im:6898523 4,604004462 6,187979584Dr.16806.1.A1_at zgc:110527 4,514592894 6,0826535Dr.6460.1.S1_at zgc:92606 6,137482592 8,27439021Dr.6309.1.A1_at wu:fj68e01 4,043025777 5,454496263Dr.3958.1.A1_at ncaph 4,538662427 6,158110154Dr.3421.1.A1_at rrbp1 5,626240996 7,643582377Dr.1809.1.A1_at calrl2 5,434457534 7,394054997Dr.11394.1.S1_at 4,139894287 5,643684622Dr.17411.1.S1_at LOC799904 7,092088233 9,753716932Dr.24938.1.S1_x_at zgc:158463 6,545587559 9,021021559Dr.13971.1.A1_at LOC559239 4,403765925 6,091030722Dr.12597.1.S1_at cyp2k6 4,520924232 6,256020623Dr.11661.1.S1_at ypel3 6,543432251 9,055356124Dr.26414.1.S1_at 3,804658728 5,298733332Dr.15069.1.S1_at zdhhc15b 9,279251355 12,95854663Dr.21263.1.S1_at asah1a 4,237319686 5,975708483Dr.14931.2.S1_at manf 5,447178417 7,717115998Dr.1593.1.A1_at 3,895048849 5,53114978Dr.9743.2.S1_a_at hmgb2b 5,96441493 8,528524082Dr.685.1.S1_at si:dkey-170l10.1 3,522122653 5,050984383Dr.14161.1.S1_at cxc46 4,46714937 6,447632035Dr.25711.1.A1_at sb:cb288 3,69096346 5,54748227Dr.3466.1.A1_at lpcat4 3,739173538 5,756678354

1312

Dr.15538.1.A1_at LOC100332348 8,140901013 12,95958835Dr.14336.1.A1_at zgc:73275 5,06255077 8,387642621Dr.18392.1.A1_at zgc:152982 4,511680342 7,481422193Dr.20850.1.S1_at fabp7a 4,495898875 7,629504727Dr.10555.1.A1_at pdia2 4,345745944 7,488389775Dr.21035.1.S1_at mhc1uaa 4,55394884 8,09269701Dr.17463.1.A1_at 4,408809634 9,401562614Dr.23469.1.S1_s_at 3,909820859 9,368173035

14

1

2

12

Suppemental Table 2: 92 common genes between CREB ChipOnChip Database and 171 Human

Homologous of differentially expressed genes between CREB-zebrafish and controls.

ABCC4 FAAH NR4A1ABCD3 FABP2 PCK1ACTN3 FABP7 PER2AHCYL1 FBXO25 PGAM2ALDOA FHL3 PKLRALOX5AP FKBP5 POP5ALPI FOS PPIBAP1M2 GADD45B PPP1R14AAQP8 GLDC PRKCDARG2 HMG20A PRLRATF3 HNF4G PVALBBAD HOXB3 RHCGBCAR1 HSD17B3 RRBP1BIRC5 HSPA1A SELPC20orf112 IL13RA2 SLC13A2CALM1 JUN SLC20A1CALR JUNB SLC25A4CEBPD LMAN1 SLC29A2CHRNA2 MAF SLC2A11CITED4 MAT2A SLC4A2CKM MFI2 SSR4CSNK1G2 MFN2 THY1CTGF MUS81 TM4SF4CYR61 MYBPC2 TNNC2DDC MYBPH TNNI2DECR1 MYL1 TOM1DUSP1 MYL3 TPM1DUSP2 NCALD TTNETS2 NEB USP2ETV5 NIT1 VAMP8F13A1 NPR3

15

1

2

3

4

5

12

Supplemental Table 3: 20 common genes among CREB-zebrafish, CREB ChipOnChip database

and Human Acute Myeloid Leukemia.

ABCC4ABCD3ALOX5APATF3CALM1CEBPDCTGFDUSP1DUSP2FOSGADD45BHOXB3HSP70JUNJUNBNPR3POP5PPP1R14APRKCDTPM1

16

1

2

34

5

6

7

12

Supplemental Table 4: Pearson correlations (r) between active CREB (CREB S133) and 4 out of

20 targets emerged by intersection among CREB-zebrafish and Human AML, quantitated by

reverse phase protein array (RPPA) in bone marrow samples from 66 pediatric patients with newly

diagnosed AML. r is significantly different from zero (p < 0.0001).

17

Pearson correlations (r)CREB S133

c-JUN S63 0.86

CREB S133

c-FOS 0.82

CREB S133

C/EBPδ 0.82

CREB S133

DUSP1 0.80

1

2

3

4

5

6

7

8

9

10

12

Name Signature Description p-value Reference

MEISSNER_BRAIN_HCP_WITH_H3K27ME3

Genes with high-CpG-density promoters (HCP) bearing the H3K27 tri-methylation

(H3K27me3) mark in brain.0.0001 10

SCHLESINGER_METHYLATED_DE_NOVO_IN_CANCER

Genes bearing H3K27me3 mark or whose promoters are bound by the polycomb

proteins SUZ12 or EED0.0001 11

MIKKELSEN_MCV6_HCP_WITH_H3K27ME3

Genes with high-CpG-density promoters (HCP) bearing the tri-methylation mark at

H3K27 (H3K27me3) in MCV6 cells0.0001 12

MEISSNER_NPC_HCP_WITH_H3K4ME3_AND_H3K27ME3

Genes with high-CpG-density promoters (HCP) bearing histone H3 trimethylation

marks at k4 (H3K4me3) and K27 ((H3K27me3) in neural precursor cells

(NPC).

0.0001 10

HALLMARK_TNFA_SIGNALING_VIA_NFKB

Genes regulated by NF-kB in response to TNF. 0.0368 MsigDB

13

TIAN_TNF_SIGNALING_VIA_NFKB

Genes modulated in HeLa cells (cervical carcinoma) by TNF via NFKB pathway. 0.0124 14

WANG_NFKB_TARGETS

Representative genes up-regulated in C2C12 cells (myoblast) lacking NFkB activity due to expression of a super repressor form of

NFKBIA.

0.0009 15

DAUER_STAT3_TARGETS_UPTop 50 genes up-regulated in A549 cells (lung cancer) expressing STAT3 off an

adenovirus vector.0.0003 16

NAGASHIMA_EGF_SIGNALING_UP

Genes up-regulated in MCF7 cells (breast cancer) after stimulation with EGF. 0.0068 17

BILD_HRAS_ONCOGENIC_SIGNATURE

Genes selected in supervised analyses to discriminate cells expressing activated

HRAS oncogene from control cells expressing GFP.

0.0015 18

SMID_BREAST_CANCER_RELAPSE_IN_LUNG_UP

Genes up-regulated in lung relapse of breast cancer. 0.0001 19

WOO_LIVER_CANCER_RECURRENCE_UP

Genes positively correlated with recurrence free survival in patients with hepatitis B-related (HBV) hepatocellular carcinoma

(HCC).

0.0001 20

Supplemental Table 5. List of the gene sets from Molecular Signature Database (MSigDB) which

highly correlates with C/EBPδ expression. P-value represents significance of Fischer's test in the

analysis Q1 vs Q2-Q4 (Related to Figure 5C).

18

1

2

3

12

Supplemental Table 6. List of the AML gene sets from MSigDB which correlates with C/EBPδ

expression. Differentially enriched Signatures in Q1 vs Q2-4, analyzed through GSEA (Gene Set

Enrichment Analysis; p<0.05; FDR<0.1).

NAME of Signature N. of genes

NES(Enrichment

score)

NOM p-val

FDR q-val

VERHAAK_AML_WITH_NPM1_MUTATED_UP 168 2.6035 0 0ROSS_AML_WITH_CBFB_MYH11_FUSION 50 2.2645 0 0

GAL_LEUKEMIC_STEM_CELL_DN 231 2.1073 0 0.0003

VALK_AML_CLUSTER_9 32 2.0987 0 0.0002

VALK_AML_WITH_FLT3_ITD 37 2.0167 0 0.0003

GENTLES_LEUKEMIC_STEM_CELL_DN 19 1.9753 0 0.0010

VALK_AML_WITH_EVI1 23 1.9028 0 0.0028

VALK_AML_CLUSTER_5 30 1.8817 0.0018

0.0028

KAMIKUBO_MYELOID_CEBPA_NETWORK 28 1.8258 0.0052

0.0053

VALK_AML_CLUSTER_15 27 1.6119 0.0211

0.0360

SUH_COEXPRESSED_WITH_ID1_AND_ID2_UP 18 1.5958 0.0228

0.0363

MUELLER_COMMON_TARGETS_OF_AML_FUSIONS_DN 27 1.5920 0.028

80.034

3

DUNNE_TARGETS_OF_AML1_MTG8_FUSION_UP 49 1.5687 0.0161

0.0375

VALK_AML_CLUSTER_13 25 1.5524 0.0324

0.0395

VALK_AML_CLUSTER_10 31 1.5400 0.0182

0.0409

ALCALAY_AML_BY_NPM1_LOCALIZATION_UP 133 1.5164 0.0037

0.0461

VALK_AML_WITH_CEBPA 34 1.4648 0.0302

0.0621

DUNNE_TARGETS_OF_AML1_MTG8_FUSION_DN 19 1.4211 0.0643

0.0803

19

1

2

3

4

12

Figure S1. CREB-plasmid and luciferase activity. (A) Human CREB cDNA fused in frame to

EGFP, or EGFP alone, under control of zebrafish pu.1 promoter, was inserted into a tol2 pDEST

vector. (B) Luciferase activity measured in embryos co-injected with βactin:EGFP (n= 3, white

bars) or βactin:EGFP-CREB plasmids (n= 5, black bars) together with the 6XCRE:LUC reporter

plasmid at 26 hpf (Unpaired Student’s t test, * p < 0.05).

20

1

2

3

4

5

6

7

12

Figure S2. Signature of the 20 selected genes. Heatmap representing the expressiom of the

selected 20 dfferentially expressed genes between CREB-zebrafish and controls (log2 q-FDR<

0.05).

21

1

2

3

4

5

6

12

Figure S3. C/ebpδ expression in CREB-zebrafish and pediatric AML. (A) C/ebpδ

immunohistochemical staining of kidney sagittal sections displays no signal on control (i and ii),

whereas positive cells are found in CREB-zebrafish (iii and iv). Scale bars: 50 µm. (B) phospho-

CREB and C/EBPδ protein expression are highly correlated in human pediatric AML. Protein

expression was quantitated by reverse phase protein array (RPPA) using bone marrow samples from

66 patients with newly diagnosed childhood AML (age range, 0-18 years). Pearson correlations (r)

for protein expression are significantly different from zero (p < 0.0001).

22

1

2

3

4

5

6

7

8

9

10

12

Figure S4. CREB and C/EBPδ expression. (A) Decreased CREB and C/EBPδ mRNA at 24 and

30h after CREB silencing in primary AML cells, compared to siRneg (RQ= 1). (B) CREB and

C/EBPδ levels normalized to β-actin showed the decrease of both proteins after 24 and 30h post

CREB silencing in primary AML cells. (C) Increased C/EBPδ mRNA at 16 and 24h after

exogenous CREB expression in primary HBM cell culture, compared to empty vector (RQ= 1). RQ:

relative quantitation. Log scale.

23

1

2

3

4

5

6

7

8

9

12

Figure S5. C/EBPδ levels in HL60 (CREB -) cell line and after silencing. (A) pCREB and CREB

levels normalized to β-actin showed lower levels of both proteins in HL60 (CREB -) cell line, compared

to HL60 (CREB+). (B) Decreased gene expression of CREB and C/EBPδ in HL60 (CREB-) cell line,

compared to HL60 (CREB+) (RQ= 1). (C) May–Grünwald Giemsa staining of HL60 (CREB+) and HL60 (CREB-) cells, following 72 h of ATRA treatment. Scale bars: 10 µm. (D) Decreased C/EBPδ mRNA

expression at 24 and 72h after C/EBPδ silencing in HL60 cell line, compared to siRneg (RQ= 1).

RQ: relative quantitation. Log scale.

24

1

2

3

4

5

6

7

8

9

10

12

Figure S6. CREB silencing and C/EBPδ overexpression. (A) CREB levels normalized to β-actin

showed a decreased CREB protein expression (0.4). (B) C/EBPδ levels normalized to β-actin

showed increased C/EBPδ levels after plasmid transfection (1.3).

25

1

2

3

4

5

6

12

Supplemental References:

1 Kwan KM, Fujimoto E, Grabher C, Mangum BD, Hardy ME, Campbell DS et al. The Tol2kit: A multisite gateway-based construction Kit for Tol2 transposon transgenesis constructs. Dev Dyn 2007; 236: 3088–3099.

2 Hsu K, Traver D, Kutok JL, Hagen A, Liu T-X, Paw BH et al. The pu.1 promoter drives myeloid gene expression in zebrafish. Blood 2004; 104: 1291–7.

3 Forrester AM, Grabher C, McBride ER, Boyd ER, Vigerstad MH, Edgar A et al. NUP98-HOXA9-transgenic zebrafish develop a myeloproliferative neoplasm and provide new insight into mechanisms of myeloid leukaemogenesis. Br J Haematol 2011; 155: 167–81.

4 Thisse B, Thisse C. Fast Release Clones: A High Throughput Expression Analysis. ZFIN Direct Data Submiss. 2004.http://zfin.org/ZDB-PUB-040907-1.

5 Accordi B, Espina V, Giordan M, VanMeter A, Milani G, Galla L et al. Functional protein network activation mapping reveals new potential molecular drug targets for poor prognosis pediatric BCP-ALL. PLoS One 2010; 5: e13552.

6 Sandoval S, Kraus C, Cho EC, Cho M, Bies J, Manara E et al. Sox4 cooperates with CREB in myeloid transformation. Blood 2012; 120: 155–165.

7 Pigazzi M, Manara E, Baron E, Basso G. MiR-34b targets cyclic AMP-responsive element binding protein in acute myeloid leukemia. Cancer Res 2009; 69: 2471–2478.

8 Pan Y-C, Li C-F, Ko C-Y, Pan M-H, Chen P-J, Tseng JT et al. CEBPD reverses RB/E2F1-mediated gene repression and participates in HMDB-induced apoptosis of cancer cells. Clin Cancer Res 2010; 16: 5770–80.

9 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402–8.

10 Meissner A, Mikkelsen TS, Gu H, Wernig M, Hanna J, Sivachenko A et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 2008; 454: 766–70.

11 Schlesinger Y, Straussman R, Keshet I, Farkash S, Hecht M, Zimmerman J et al. Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer. Nat Genet 2007; 39: 232–236.

12 Mikkelsen TS, Hanna J, Zhang X, Ku M, Wernig M, Schorderet P et al. Dissecting direct reprogramming through integrative genomic analysis. Nature 2008; 454: 49–55.

13 Liberzon A, Subramanian A, Pinchback R, Thorvaldsdóttir H, Tamayo P, Mesirov JP. Molecular signatures database (MSigDB) 3.0. Bioinformatics 2011; 27: 1739–1740.

14 Tian B, Nowak DE, Jamaluddin M, Wang S, Brasier AR. Identification of direct genomic targets downstream of the nuclear factor-κB transcription factor mediating tumor necrosis factor signaling. J Biol Chem 2005; 280: 17435–17448.

15 Wang H, Hertlein E, Bakkar N, Sun H, Acharyya S, Wang J et al. NF-kappaB regulation of YY1 inhibits skeletal myogenesis through transcriptional silencing of myofibrillar genes. Mol Cell Biol 2007; 27: 4374–4387.

26

1

234

56

789

1011

121314

1516

1718

192021

2223

2425

262728

2930

3132

333435

363738

12

16 Dauer DJ, Ferraro B, Song L, Yu B, Mora L, Buettner R et al. Stat3 regulates genes common to both wound healing and cancer. Oncogene 2005; 24: 3397–408.

17 Nagashima T, Shimodaira H, Ide K, Nakakuki T, Tani Y, Takahashi K et al. Quantitative transcriptional control of ErbB receptor signaling undergoes graded to biphasic response for cell differentiation. J Biol Chem 2007; 282: 4045–56.

18 Bild AH, Yao G, Chang JT, Wang Q, Potti A, Chasse D et al. Oncogenic pathway signatures in human cancers as a guide to targeted therapies. Nature 2006; 439: 353–357.

19 Smid M, Wang Y, Zhang Y, Sieuwerts AM, Yu J, Klijn JGM et al. Subtypes of breast cancer show preferential site of relapse. Cancer Res 2008; 68: 3108–3114.

20 Woo HG, Park ES, Cheon JH, Kim JH, Lee JS, Park BJ et al. Gene expression-based recurrence prediction of hepatitis B virus-related human hepatocellular carcinoma. Clin Cancer Res 2008; 14: 2056–2064.

27

12

345

67

89

101112

13

14

12