Supporting Materials and Methods

Supporting Materials and Methods Immunohistochemistry Immunohistochemistry for GATAD1 was performed on paraffin sections of TMAs using anti-GATAD1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The extent of GATAD1 nuclear staining was assessed by H-score. For semi-quantitative analysis of immunoreactivity of GATAD1, H-score was used in this study. Briefly, more than 500 tumor cells were counted in each case, and the H-score was subsequently generated by adding the percentage of strongly stained nuclei (3×), the percentage of moderately stained nuclei (2×), and the percentage of weakly stained nuclei (1×), giving a possible range of 0–300 (1). Cases that were found to have an H-score more than 27.5 were noted as GATAD1 high expression, according to the Receiver Operating Characteristic (ROC) curve analysis. Moreover, the cutoff value for GATAD1 positive expression was defined as an H-score more than 10. Immunohistochemistry for GATAD1 and Ki67 was also performed on paraffin slides of mouse xenograft tumors using anti-GATAD1 and anti-Ki67 antibodies (Abcam, Cambridge, UK).  

RNA extraction and real-time PCR analyses Total RNA was extracted using TRIzol and transcribed into cDNA using a High Capacity cDNA Kit (Applied Biosystems, Foster City, CA). For quantitative PCR analysis, aliquots of cDNA were amplified using SYBR® Premix Ex Taq™ II (Takara Bio Inc, Japan) on LightCycler® 480 Instrument (Roche Diagnostics, Switzerland). Each sample was tested in triplicate. ΔΔCT method was employed to    

1

determine the fold change in gene expression level. ΔCT method was employed to determine the relative expression levels of corresponding genes. The sequences of primers used are listed in Supporting Table S5.

Western blot Protein lysates from cell lines and tissues were prepared using protease inhibitor cocktail (Roche)–containing radioimmunoprecipitation assay (RIPA) buffer. Protein concentration was determined by the DC

protein

assay

method

of

Bradford (Bio-Rad, Hercules, CA). Proteins were separated on sodium dodecyl sulfate-polyacrylamide gel

electrophoresis

nitrocellulose membranes (GE

Healthcare,

(SDS-PAGE) and transferred onto Piscataway,

NJ).

Blots

were

immunostained with primary antibodies at 4℃ overnight and secondary antibody at room temperature for 1 hour. Proteins were visualized using ECL Plus Western Blotting Detection Reagents (GE Healthcare). The antibodies used in this study are listed in Supporting Table S6.

Construction

of

gene

expression

plasmid

and

establishment

of

stable

GATAD1-expressing cells The full-length open reading frame sequence of GATAD1 was obtained by RT-PCR amplification of normal human liver cDNA. The PCR aliquots were subcloned into the lenti-viral vector pLVX-IRES-Puro and then verified by DNA sequencing. Overexpression of GATAD1 in LO2 or HepG2 cells were performed by lenti-virus    

2

mediated transfection. psPAX2, pMD2.G and pLVX-GATAD1 or empty vector pLVX were co-transfected into HEK 293T cells using lipofectamine 2000 (Life Technologies, Carlsbad, CA). Two days after transfection, the cell supernatant containing recombinant lentiviral vectors was collected and transduced into LO2 and HepG2 cells. After 48 hours, stable transfections were selected with puromycin antibiotics for 2 weeks.

The full-length open reading frame sequence of PRL3 was obtained by RT-PCR amplification of normal human liver cDNA. The PCR aliquots were subcloned into the pCMV6 vector and then verified by DNA sequencing.

Lenti-virus-mediated shRNA targeting GATAD1 Knock-down Knock-down GATAD1 expression in SK-Hep1 and HepG2 cell lines was performed by a lenti-virus-mediated shRNA targeting GATAD1. Both sh-Control and sh-GATAD1 (shGATAD1#1: 5’-GAG UCA GUU UCC ACU AUA A-3’; shGATAD1#2: 5’-GAC CAG UAU UGC GAG AAG A-3’) cells were selected with puromycin for 2 weeks after transfection for 48 h. SK-Hep1 and HepG2 cells were transfected with 50 nM PRL3 siRNA (siPRL3#1: 5’-GGC AAG GUA GUG GAA GAC U-3’; siPRL3#2: 5’-GAG GUG AGC UAC AAA CAC A-3’) (Shanghai genephama Co., Ltd, China) or control siRNA (Shanghai genephama Co., Ltd, China) using lipofectamine 2000 (Life Technologies).

   

3

Colony formation assay For overexpression assay, LO2 and HepG2 cells (103 cells/well), which were stablely transfected with lenti-PLVX–GATAD1 or lenti-empty vector were plated in a 6-well plate. For knockdown assay, SK-Hep1 and HepG2 cells were stably transfected with lenti-sh-GATAD1 or lenti-sh-Control. Meanwhile, LO2 and HepG2 cells were transfected with siPRL3 or control siRNA. After culturing for 14–21 days, cells were fixed with methanol and stained with crystal violet solution. Colonies with > 50 cells per colony were counted. All experiments were conducted three times in triplicates.

Cell viability assay Cell viability was measured by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay (Promega, Fitchburg, WI). The experiments were performed three times independently. Results were shown as the means ± SD.

Cell cycle analysis LO2 and HepG2 cells that were stably transfected with GATAD1 or empty vector were plated in a 6-well plate, while SK-Hep1 and HepG2 cells were transfected with siGATAD1 or si-Control. After 48 h of transfection, the cells were fixed in ice-cold 70% ethanol-phosphate-buffered saline for 24 h before staining with 50 μg/ml propidium iodide (BD Biosciences, Franklin Lakes, NJ). The cells were sorted by BD Accuri™ C6 (BD Biosciences), and cell cycle distributions were analyzed using the ModFitLT 4.1 software (Verity Software House, Topsham, ME). All    

4

experiments were conducted three times in triplicates.

Apoptosis Cell apoptosis of the GATAD1 overepression cells (LO2 and HepG2) and GATAD1 knockdown cells (SK-Hep1 and HepG2) was determined by staining cells with Annexin V and 7-amino-actinomycin D (7-AAD) (BD Biosciences) with subsequent flow cytometry analysis. Cell populations were counted as viable (Annexin V-negative, 7-AAD-negative), early apoptotic (Annexin V-positive, 7-AAD-negative), late apoptotic (Annexin V-positive, 7-AAD-positive), or necrotic (Annexin V-negative, 7-AAD-positive). The experiments were conducted three times in triplicates. In addition, terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay (Roche) was employed for apoptosis measurement of nude mice tumor biopsies. Nuclei with clear brown staining were regarded as TUNEL-positive apoptotic cells. The apoptosis index was calculated as the percentage of TUNEL-positive nuclei after counting at least 1000 cells.

Migration and invasion assays For migration and invasion assays, migration transwell chambers and matrigel-coated chambers (Becton Dickinson, Waltham, MA, USA) were used. Briefly, 1×104 cells were seeded into the upper chamber in serum-free culture medium. The lower chamber was filled with completed medium with 10% FBS. After 24h for migration assay and 48h for invasion assay, cells that have migrated or invaded through the    

5

membrane were stained with crystal violet and counted.

In vivo tumorigenicity assay The LO2 cells (5 × 106) with or without GATAD1 transfection were subcutaneously injected into the right and left flanks of the 4-week-old male Balb/c nude mice. For knockdown assay, HepG2 cells that stably transfected with lenti-sh-GATAD1 or lenti-sh-Control were used. Tumor size was measured every 2 days for 2 weeks using a caliper. Tumor volume (mm3) was estimated by measuring the longest and shortest diameter of the tumor (Formula: Volume = 0.5 × Length × 2 × Width). Mice were sacrificed at 2 weeks after injection. Tumors were excised and weighed. The excised tissues were either fixed in 10% neutral-buffered formalin or snap frozen in liquid nitrogen. Tumor sections from paraffin-embedded blocks were used for histologic examination. All animal studies were performed in accordance with guidelines approved by the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong.

An orthotopic HCC mouse model was established using HepG2. HepG2 cells (5106 cells in 0.1 ml PBS) transduced by shGATAD1 lentivirus and negative control vector-lentivirus (shNC) were injected subcutaneously into the right and left dorsal flank of 4-week-old male Balb/c nude mice, respectively. Subcutaneous tumors were harvested after two weeks and cut into 1.0 mm3 pieces. One piece of the tumor was then implanted into the left liver lobe in a separate group of 6-week old nude mice (n    

6

= 5/group). Four weeks after tumor implantation, the mice were sacrificed and examined. The livers and lungs were dissected and paraffin embedded, and the sections were stained with hematoxylin and eosin. Tumors in the liver and metastatic tumors in the lungs were counted in a blinded manner. All animal experimental procedures were approved by the Animal Ethics Committee of the Chinese University of Hong Kong.

Four-week-old Balb/c nude mice were inoculated subcutaneously with 4×105 HepG2 cells into the right flanks. Once palpable tumors formed, mice were randomized into BR-1 treatment group or vehicle group. The mice in the treatment group were treated with 50 mg/kg BR-1 (BR-1 was first dissovled in DMSO at 25mg/ml, then the drug was diluted in olive oil. The final concerntration of the drug that used for injection was 3.125 mg/ml.) three times per week by i.p. injcetion. Tumor measurements were reorded every two days. All animal experimental procedures were approved by the Animal Ethics Committee of the Chinese University of Hong Kong.

Immunofluorescence Ectopic expression of LO2 and HepG2 cells were seeded in 12-well plates and fixed in 4% paraformaldehyde (Sigma, St. Louis, MO) for 30 min. Cells were then permeabilized with PBS containing 0.1% Triton X-100 for 3 min and incubated with a 5 mg/ml BSA blocking solution. Cells were incubated with GATAD1 antibody (1:100, Santa Cruz Biotechnology), followed by incubation with a secondary antibody.    

7

Chromosomes were stained with DAPI in PBS for 5 min. The immunofluorescence images were taken with a fluorescence microscope (Olympus, Tokyo, Japan).

GATAD1 transcription factor binding motif (TFBS) prediction GATAD1 transcription factor binding motif (TFBS) prediction ChIP-seq of GATA1D has already been completed in Hela (Kyoto) Cells which are derived from http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE20303. In particular, the ChIP-seq file has been processed into peak file. The genome-wide binding locations of GATAD1 in Hela (Kyoto) cells can be accurately estimated from the peak file. To create and estimate the canonical TFBS pattern of GATAD1, we retrieved the corresponding peak sequences in hg 19. Based on the fasta file, we ran the state-of-the-art TFBS modeling software DREME. From the DREME output, it appears that there are 6 TFBS patterns (Supporting Fig. S7). To understand them, we further ran TomTom to check if there is any match to existing TFBS patterns of known TF. Since GATAD1 has a zinc finger domain, the most likely TFBS pattern for GATAD1 is the 3rd one (CCCMGCCC) by TomTom. The remaining 5 TFBS patterns are likely to be the TFBSs of cofactors.

RNA-sequencing The total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. All samples had RIN values above 6.8. Sequencing libraries were generated using IlluminaTruSeq™ RNA Sample Preparation Kit (Illumina, San    

8

Diego, CA), following manufacturer’s recommendations. The libraries were sequenced on an Illumina HiSeq 2000 platform as per manufacturer’s instructions.

Dual-luciferase reporter assay To investigate the signaling pathways modulated by GATAD1, a series of signaling pathway luciferase reporters were examined in GATAD1-transfected LO2 and HepG2 cells, including nuclear factor (NF)-κB-luc (5 × NF-κB binding sites), p53-luc (14 × p53 binding sites), AP1-luc (7 × AP1 binding sites), SRE-luc (5 × SRE binding sites), and superTOP (4 × TCF binding sites) and forkhead responsive element (FHRE)-luc (a gift from Michael Greenberg from Harvard Medical School (Addgene plasmid # 1789). The cell lines (LO2 and HepG2) stably transfected with pLVX-GATAD1 or pLVX (1 x 105 cells/well) in 24-well plates and were cotransfected with luciferase report plasmid (0.2 μg/well) and pRL-cyto-megalovirus (pCMV) vector (5 ng/well) using lipofectamine 2000 (Life Technologies). Cells were harvested 48 hours post-transfection and luciferase activities were analyzed by the dual-luciferase reporter assay system (Promega).

Co-immunoprecipitation assay LO2 and HepG2 cells were transfected with pCMV6-PRL3 expressing vector or pCMV6 empty vector. After

48 hours

post-transfection, total proteins were

extracted by CytoBuster Protein Extraction Reagent (Novagen, Darmstadt, Germany). For each immunoprecipitation, 300 μg of precleared cell lysate and 30 μl of    

9

protein G PLUS-Agarose (Santa Cruz Biotechnology) were used for overnight incubation at 4 ℃ . The immunoprecipitated proteins were mixed with 2 × SDS-PAGE loading buffer and boiled at 95℃ for 10 minutes. The proteins were separated by SDS-PAGE and validated by Western blot.

Chromatin immunoprecipitation (ChIP) assay A total of 1 × 107 LO2 or HepG2 cells stably transfected with GATAD1-myc were crosslinked with 1% formaldehyde for 10 and quenched by glycine. After cell lysis,

minutes at room temperature

the chromatin was

fragmented into

200–600 bp by sonication and protein-DNA complexes were immunoprecipitated (IP) by 2 μg anti-myc-tag antibody (Cell Signaling, Danvers, Massachusetts) or anti-IgG antibody (Abcam) Dynal magnetic bead (Millipore, Jaffrey, NH) mix on rotator at 4°C overnight. After washing and reversal of

crosslinks, the IP and

input DNA were purified. For target gene validation, PCR primers targeting a region of the putative binding site were designed to detect IP and input DNA. 2 μl IP and 1% input DNA were used as a template for conventional PCR assay. For quantitative ChIP-PCR, equal amounts of IP and diluted input DNA were used for SYBR Green–based detection (Takara). The sequences of primers used are listed in Supporting Table S5.

   

10 

Figure S1 Migration 150 150 100 100

5050

Invasion

250 250

Vector

Relative invasion ability (%)

LO2 GATAD1

Slug β-actin

GATAD1

Vector N-cadherin

GATAD1

HepG2

LO2

Vector

C

HepG2

P < 0.01

Vector

200 200

200 200

200 200

GATAD1

100 100 50 50

P < 0.001

400 400

00

150 150

00

600 600

Vector GATAD1

Relative invasion ability (%)

00

B

GATAD1

Vector GATAD1 P < 0.05

150 150 100 100 50 50

00

Vector GATAD1

Vector GATAD1

H&E

D

Number of metastasis lesions

GATAD1

Vector

HepG2/shNC

Relative migration ability (%)

Vector

HepG2

P < 0.05

Relative migration ability (%)

200 200

LO2

HepG2/shGATAD1

A

88 66 44 P < 0.05 22 00

shNC shGATAD1

Fig. S1. GATAD1 promotes the migration and invasion abilities in HCC in vitro and in vivo. (A) Representative images of migration transwell assay revealed that ectopic expression of GATAD1 promoted HCC cells migration. (B) Representative images of matrigel invasion assay revealed that ectopic expression of GATAD1 promoted HCC cells invasion. (C) Western blot analysis revealed that ectopic expression of GATAD1 increased expression of Slug and N-cadherin. (D) Representative macroscopic appearances of lung metastasis are shown. Representative hematoxylin-eosin stained images of the lungs were shown in the right panel. silencing of GATAD1 in HepG2 cells significantly reduced the number of metastatic lesions in the lungs (P < 0.05). Data was expressed as mean ± S.D. Student's t-test was performed.    

11 

Figure S2 LO2 P < 0.05

22

Relative mRNA level

33

Vector GATAD1

P < 0.05

11 00

B

BIRC7

ICAM5 LO2

88

HepG2

66 44

BIRC7 (-1126~-1119) ICAM5 (-397~-390) ICAM5 (-1111~-1104) ACTB

P < 0.001

Vector GATAD1

P < 0.05

22 00

C

HepG2

BIRC7 mRNA expression

Relative mRNA level

A

BIRC7

ICAM5

BIRC7 50 50

r = 0.790 P < 0.001

40 40

30 30

20 20 15 15

20 25 30 35 40 20 25 30 35 40 GATAD1 mRNA expression

Fig. S2. The influence of GATAD1 on potential target genes of BIRC7 and ICAM5. (A) GATAD1 induced the mRNA levels of BIRC7 and ICAM5 by qPCR. (B) ChIP-PCR confirmed the enrichment of GATAD1 in the promoters of BIRC7 and ICAM5. (C) The correlation betwteen GATAD1 mRNA level and BIRC7 mRNA level was confirmed in 50 cases of HCC tumor tissues.

   

12 

Figure S3 LO2 ns

Vector GATAD1

ns

1.0 1.0

0.5 0.5

0.0 0

AKT1

1.5 1.5

Relative mRNA level

Relative mRNA level

1.5 1.5

AKT2

HepG2

Vector GATAD1

ns

ns

AKT1

AKT2

1.0 1.0

0.5 0.5

0.0 0

Fig. S3. GATAD1 did not increase the mRNA level of AKT1 and AKT2. Real-time PCR confirmed that overexpression of GATAD1 in LO2 and HepG2 cell lines did not increase the mRNA level of AKT1 and AKT2. ns, not significant.

   

13 

Figure S4 siPRL3

siNC

siPRL3

siNC

SK-Hep1 HepG2

PRL3 p-Akt (Thr450) p-Akt1 (Ser473) Akt1 p-Akt2 (Ser474) Akt2 β-actin

Fig. S4. PRL3 knockdown reduced the protein levels of phospho-Akt (p-Akt), phospho-Akt1 (p-Akt1) and phospho-Akt2 (p-Akt2) by Western blot.

   

14 

Figure S5

GATAD1

p-mTOR

p-p38

mTOR

GATAD1

Vector

GATAD1

HepG2 GATAD1

LO2

Vector

B

GATAD1

HepG2 GATAD1

Vector

LO2

Vector

A

p38

p-p70S6K(Thr389)

β-actin

p-4E-BP1 β-actin

Fig. S5. GATAD1 activated mTOR and p38MAPK signaling pathway. (A) phospho-mTOR as well as the downstream effector of mTOR, phospho-p70 S6 Kinase (Thr389) and phospho-4E-BP1 were elevated in GATAD1 overexpression LO2 and HepG2 cells by Western blot. (B) The phospho-p38MAPK was also increased in GATAD1 overexpression cells.

   

15 

Figure S6 Body Weight (g)

30 30

n.s.

20 20

10 10

00

Vehicle

BR-1

Fig. S6. Treatment with BR-1 in nude mice had on obvious effect on body weight.

   

16 

Figure S7

Fig. S7. Six putative transcription factor binding site (TFBS) patterns of GATAD1. The most likely TFBS pattern for GATAD1 was the 3rd one (CCCMGCCC).

   

17 

Table S1. Univariate and multivariate Cox regression analyses of potential poor prognostic factors in HCC  Variables Age Gender Male Female Grade Low Moderate/High TNM Ⅰ/Ⅱ Ⅲ/Ⅳ Size ≥ 5 cm < 5 cm Multiple Tumor Yes No GATAD1 expression High Low    



Univariate analysis RR(95%CI)

P value



Multivariate analysis RR(95%CI)

P value

1.155 (0.632 to 2.113)

0.640

1.035 (0.548 to 1.953)

0.916

0.637 (0.271 to 1.501) 1

0.303

0.728 (0.306 to 1.733) 1

0.474

1.817 (0.848 to 3.891) 1

0.124

0.174 (0.097 to 0.311) 1

＜0.0001

0.249 (0.122 to 0.507)

＜0.0001

2.568 (1.439 to 4.582) 1

0.001

1.346 (0.681 to 2.661) 1

0.393

3.955 (2.205 to 7.093) 1

＜0.0001

1.954 (0.955 to 4.000) 1

0.067

1.867 (0.999 to 3.489) 1

0.043  

1.981 (1.056 to 3.716) 1

0.033

18 

 

 

Table S2. Univariate and multivariate Cox regression analyses of potential poor prognostic factors in stage I/II HCC  Variables Age Gender Male Female Grade Low Moderate/High TNM Ⅰ Ⅱ Size ≥ 5 cm < 5 cm Multiple Tumor Yes No GATAD1 expression High Low

   

Univariate analysis RR(95%CI) P value 1.674 (0.751 to 3.729) 0.207 1.023 (0.384 to 2.727) 1

0.964

1.805 (0.619 to 5.263) 1

0.279

0.321 (0.146 to 0.708) 1

0.005

1.008 (0.421 to 2.413) 1

0.986

1.532 (0.458 to 5.128) 1

0.489

4.195 (1.440 to 12.224) 1

0.009  

19 

 

 

Multivariate analysis RR(95%CI) P value 2.573 (1.122 to 5.900) 0.026 1.309 (0.490 to 3.501) 1

0.591

0.222 (0.098 to 0.506) 1

＜0.0001

5.577 (1.891 to 16.442) 1

0.002  

Table S3. A list shows upregulated candidate genes in both LO2 and HepG2 GATAD1-overexpression cell lines ENSEMBL ID

Gene Name

ENSG00000002726 amiloride binding protein 1 (amine oxidase (copper-containing)) ENSG00000054938 chordin-like 2 ENSG00000064225 ST3 beta-galactoside alpha-2,3-sialyltransferase 6 ENSG00000065361 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) ENSG00000080644 cholinergic receptor, nicotinic, alpha 3 ENSG00000092068

solute carrier family 7 (cationic amino acid transporter, y+ system), member 8

ENSG00000101197 baculoviral IAP repeat-containing 7 ENSG00000105219 cyclin N-terminal domain containing 2 ENSG00000105376 intercellular adhesion molecule 5, telencephalin ENSG00000106113 corticotropin releasing hormone receptor 2 ENSG00000115255 receptor accessory protein 6 ENSG00000115290 growth factor receptor-bound protein 14 ENSG00000115596 wingless-type MMTV integration site family, member 6 ENSG00000117643 mannosidase, alpha, class 1C, member 1 ENSG00000125266 ephrin-B2 ENSG00000133216 EPH receptor B2 ENSG00000135744 angiotensinogen (serpin peptidase inhibitor, clade A, member 8) ENSG00000152377

sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1

ENSG00000155158 tetratricopeptide repeat domain 39B ENSG00000162843 WD repeat domain 64 ENSG00000162894 Fas apoptotic inhibitory molecule 3 ENSG00000163354 DC-STAMP domain containing 2 ENSG00000164099 protease, serine, 12 (neurotrypsin, motopsin) ENSG00000164638

solute carrier family 29 (nucleoside transporters), member 4; similar to solute carrier family 29 (nucleoside transporters), member 4

ENSG00000166924 chromosome 7 open reading frame 51

   

20 

ENSG00000167183 ATPase family, AAA domain containing 4 ENSG00000168481 leucine-rich repeat LGI family, member 3 ENSG00000169902 tyrosylprotein sulfotransferase 1 ENSG00000170379

family with sequence similarity 115, member C; family with sequence similarity 115, member D (pseudogene)

ENSG00000172346 cold shock domain containing C2, RNA binding ENSG00000178752 family with sequence similarity 132, member B ENSG00000178821 transmembrane protein 52 ENSG00000181029 trafficking protein particle complex 5 ENSG00000184489 protein tyrosine phosphatase type IVA, member 3 ENSG00000185885 interferon induced transmembrane protein 1 (9-27) ENSG00000186832 keratin 16; keratin type 16-like ENSG00000187486 potassium inwardly-rectifying channel, subfamily J, member 11 ENSG00000198576 activity-regulated cytoskeleton-associated protein ENSG00000213937 claudin 9                                                        

21 

Table S4. A list shows 16 candidate genes were significantly upregulated in HCC tumor tissues from TCGA dataset ENSEMBL ID

Gene Name

ENSG00000064225 ST3 beta-galactoside alpha-2,3-sialyltransferase 6 ENSG00000065361 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) ENSG00000080644 cholinergic receptor, nicotinic, alpha 3 ENSG00000101197 baculoviral IAP repeat-containing 7 ENSG00000105219 cyclin N-terminal domain containing 2 ENSG00000105376 intercellular adhesion molecule 5, telencephalin ENSG00000106113 corticotropin releasing hormone receptor 2 ENSG00000115596 wingless-type MMTV integration site family, member 6 ENSG00000125266 ephrin-B2 ENSG00000133216 EPH receptor B2 ENSG00000152377

sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1

ENSG00000163354 DC-STAMP domain containing 2 ENSG00000164638

solute carrier family 29 (nucleoside transporters), member 4; similar to solute carrier family 29 (nucleoside transporters), member 4

ENSG00000168481 leucine-rich repeat LGI family, member 3 ENSG00000184489 protein tyrosine phosphatase type IVA, member 3 ENSG00000187486 potassium inwardly-rectifying channel, subfamily J, member 11        

   

22 

Table S5. DNA sequences of primers used in this study Primer name

Sequence (5'-3')

RT-PCR GATAD1-F

TGAAAAATCCCATCAAAGCTCCTG

GATAD1-R

TGCTGCACTCTTCTCGCAAT

β-actin-F

CATCCACGAAACTACCTTCAACTCC

β-actin-R

GAGCCGCCGATCCACACG

PRL3-F

GGTTCTGCTACTCCACTTGCT

PRL3-R

AGAAGTACGGGGCTACCACTG

BIRC7-F

GCTCTGAGGAGTTGCGTCTG

BIRC7-R

CACACTGTGGACAAAGTCTCTT

ICAM5-F

CAGAGGGGTTTGCGTTGGTT

ICAM5-R

GAAAGTGCGAATGAGCCCAC

AKT1-F

GCACAAACGAGGGGAGTACA

AKT1-R

AAGGTGCGTTCGATGACAGT

AKT2-F

ACCACAGTCATCGAGAGGACC

AKT2-R

GGAGCCACACTTGTAGTCCA

ChIP-PCR PRL3(-3817~-3810)-F

GCCGCATTTCCATTTCCAAAG

PRL3(-3817~-3810)-R

CGCCCTCCCTAGAACCTC

PRL3(-985~-978)-F

GGCTGGAGAGTCTGCTAACA

PRL3(-985~-978)-R

CTACGGAAGGGCTGGGAG

BIRC7(-1126~-1119)-F

CCAGAACAGTCACCAGAG

BIRC7(-1126~-1119)-R

CACCACACTTTCACACCT

ICAM5(-1111~-1104)-F

GGGTTCATGGTATGGCTTTCCT

ICAM5(-1111~-1104)-R

GAGTTCTCGCTCCACAGTTGTT

ICAM5(-397~-390)-F

CATCCAGTGCCTCCCAAATCTC

ICAM5(-397~-390)-R

TGCCCTTCCCAAACCCCTA

ACTB-F

TGTTTGAACCGGGCGGAG

ACTB-R

TAAAAGGCAAACACTGGTCGG

   

23 

Table S6. A list shows the antibodies used Antibody name

Company

Catalog No.

Dilution

GATAD1

Santa cruz

sc-81092

1:500

β-actin

Santa cruz

sc-47778

1:2,000

Cyclin-D1

Cell Signaling Technology

#2922

1:1,000

Cyclin-D3

Cell Signaling Technology

#2936

1:1,000

CDK4

Cell Signaling Technology

#12790

1:1,000

p21

Santa cruz

sc-6246

1:500

p27

Cell Signaling Technology

#2552

1:1,000

Cleaved caspase-9

Cell Signaling Technology

#9501

1:1,000

Cleaved caspase-7

Cell Signaling Technology

#9491

1:1,000

Cleaved caspase-3

Cell Signaling Technology

#9661

1:1,000

Cleaved PARP

Cell Signaling Technology

#5625

1:1,000

Caspase-9

Cell Signaling Technology

#9508

1:1,000

Caspase-7

Cell Signaling Technology

#9492

1:1,000

Caspase-3

Cell Signaling Technology

#9665

1:1,000

PARP

Cell Signaling Technology

#9532

1:1,000

phospho-Akt (Thr450)

Cell Signaling Technology

#9267

1:1,000

phospho-Akt1

Cell Signaling Technology

#9018

1:1,000

Cell Signaling Technology

#8599

1:1,000

Akt1

Cell Signaling Technology

#2938

1:1,000

Akt2

Cell Signaling Technology

#3063

1:1,000

phospho-GSK3β

Cell Signaling Technology

#9336

1:1,000

GSK3β

Cell Signaling Technology

#9315

1:1,000

phospho-mTOR

Santa cruz

sc-101738

1:500

mTOR

Cell Signaling Technology

#2972

1:1,000

phospho-p38 MAPK

Cell Signaling Technology

#4511

1:1,000

p38 MAPK

Proteintech

14064-1-AP

1:500

phospho-p70S6K

Cell Signaling Technology

#9862

1:1,000

Cell Signaling Technology

#9862

1:1,000

(Ser473) phospho-Akt2 (Ser474)

(Thr389) Phosphor-4E-BP1    

24 

Slug

Cell Signaling Technology

#9585

1:1,000

N-cadherin

Cell Signaling Technology

#4061

1:1,000

PTEN

Santa cruz

sc-7974

1:500

Phosphotyrosine

Abcam

ab190824

1:1,000

PRL3

Abcam

ab82568

1:500

     

Reference 1.

Ishibashi H, Suzuki T, Suzuki S, Moriya T, Kaneko C, Takizawa T, Sunamori M,

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