Supplementary Text

siRNA oligos and lentivirus vector generation

Multiple siRNA duplex oligonucleotides were tested for their specificity of PDEF

inhibition and a lentivirus encoding the most efficient PDEF siRNA was generated. Three

different siRNAs (Dharmacon) for the human PDEF gene were designed and evaluated

for their abilities to reduce expression of Flag-PDEF-wt in HEK 293 cells as described

before (Zerbini) in order to identify the most effective siRNA against PDEF. Briefly, the

Flag-PDEF-wt expression vector was transfected into the cells using LT1 transfection

reagent (Mirus). Chemically synthesized RNA oligos were annealed, deprotected, and desalted as recommended by the manufacturer (Dharmacon). 50µM of RNA duplexes were transfected into the cells using TKO transfection reagent (Mirus). extracts were collected 48 hours post-transfection and analyzed by Western Blot using anti-Flag antibody (Sigma). The sense-strand sequence below (a complementary oligonucleotide was synthesized for each) is the one chosen among the three oligos tested and was

effective in silencing Flag-PDEF-wt gene expression. The PDEF siRNA is 5'- -

AAGCUGCUCAACAUCACCGCA 3’; The siRNA oligo for the GFP gene (5’-

GCAAGCTGACCCTGAAGTTC-3’) as previously described (1) was used as control in

all transfection experiments.

Production and evaluation of lentiviral vectors

Vesicular stomatitis virus G envelope protein-pseudotyped lentiviruses were prepared

and purified as previously described (1, 2). Vector concentrations were analyzed by immunocapture p24-gag ELISA (Alliance, DuPont/NEN) (3). We tested the specificity of

all lentivirus vectors by real time PCR and Western blotting.

Generation of cell lines stably expressing PDEF siRNA PDEF

PC-3 and LNCaP cells were infected with the PDEF siRNA or GFP siRNA lentivirus at

MOI 10. Twenty-four hours post-infection, the media were replaced and cells incubated for an additional 48 hours. PC-3 and LNCaP cells infected with the PDEF siRNA lentivirus were washed with PBS, resuspended in serum free medium at 5x107 cells/ml and sorted based on the GFP signal using the MoFlow cell sorter (Cytomation, Fort

Collins, CO). GFP positive cells were collected in medium containing 10% fetal bovine serum and allowed to grow for 48 hours. Protein extracts from the sorted cells were collected and analyzed by Western Blot using anti-PDEF antibody. These cell lines were used in all experiments.

Stable Transfection

The PDEF-wt and PDEF-DN expression constructs were made by subcloning the full- length wild type PDEF cDNA or a deletion mutant of PDEF containing the Pointed domain and the Ets domain, but lacking the N-terminal transactivation domain into the

constitutive mammalian expression vector pCDNA 5’-Flag (Invitrogen) to generate the pCDNA-PDEF-wt and pCDNA-PDEF-DN (FLAG-tagged) constructs. Briefly, PDEF-wt, and PDEF-DN were PCR-amplified using Hi-Fidelity Taq polymerase (Invitrogen) and inserted into the BamHI and XhoI sites of the 5'-FLAG-pcDNA3.1 vector. 1 x 106 PC-3 cells or LNCaP cells were transfected with 10 ug of each expression vector DNA using LipofectAMINE plus reagent (Invitrogen) as described previously (4). Two days after

transfection, stably transfected cells were selected by adding G418 (Invitrogen) at 500

ng/ML concentration into the culture medium. Stably transfected cell lines were

established from the pooled G418 resistant cells as PC-3 PDEF-wt, PC-3 PDEF-DN, and

PC-3 Flag, and LNCaP PDEF-wt, LNCaP PDEF-DN, and LNCaP Flag.

Construction of Adenovirus and Adenoviral Infection

The adenovirus encoding the Flag-PDEF gene was generated using the ADENO-X

system from CLONTECH Laboratories, Inc., USA as described previously (5). Briefly,

the fragment encoding the Flag-PDEF gene was obtained from the pCDNA3-PDEF-flag

plasmid. Flag-tagged PDEF was subcloned into the pShuttle vector (3.9kb), creating an independent expression cassette. The cassette was transferred to pAdeno-X (32.6 Kb) containing adenoviral DNA by means of in vitro ligation at the sole restriction sites I-Ceu

I and PI-Sce I. The ligation was digested by Swa I in order to linearize non-recombinant

(self-ligated) pAdeno-X DNA. The cosmids were then used to transform electrocompetent DH5α (Invitrogen) bacterial cells, using standard molecular biology techniques. Recombinant adenoviral DNA was purified, linearized with PacI, and transfected into the 293A cells using Lipofectin plus reagent (Invitrogen). Recombinant

adenoviruses were plaque purified and screened for the expression of PDEF by Western blot. MDA-MB-231 cells were infected at 100 MOI with either control vector (Ad-flg) or

Ad-PDEF for 16 h in 5% serum (5). Infected MDA-MB-231 cells were used for migration and invasion assays.

Real Time PCR

Total RNA was harvested by using QIASHREDDER (Qiagen, Valencia, CA) and the

RNeasy minikit (Qiagen). Real-time PCR was performed as described (6).

The primers used for real time PCR are:

Vimentin sense 5’GGCTCAGATTCAGGAACAGC3’

Vimentin antisense 5’GCTTCAACGGCAAAGTTCTC3’

Cytokeratin18 sense 5’CTGGAGACCGAGAACCGGA3’

Cytokeratin18 antisense 5’TCCGAGCCAGCTCGTCAT3’

SNAIL2 sense 5’GACCACAGTGGCTCAGA3’

SNAIL2 antisense 5’GTGTCCTTGAAGCAACCA3’

E-cadherin sense 5’GAGGAGAGCGGTGGTCAAAG3’

E-cadherin antisense 5’GCGTTCCTGTTCCACTCATAGGAGG3’

PDEF sense 5’GTCCGCCTTCTACCTCTCCT 3’

PDEF antisense 5’ CAGGTGAAGTCCGCTCTTTC 3’

GAPDH sense 5’ CAAAGTTGTCATGGATGACC 3’

GAPDH antisense 5’ CCATGGAGAAGGCTGGGG 3’

Western Blot analysis

Whole cell lysates were prepared in lysis buffer (20mM Tris pH7.4, 150mM NaCl, 1mM

EDTA, 1mM EGTA, 1% Triton X100, 2.5mM sodium pyrophosphate, 1mM β- glycerolphosphate, 1mM Na3VO4, 1µg/ml leupeptin and 1mM PMSF). Thirty milligrams of protein were electrophoresed in a 10% polyacrylamide-SDS gel. were electroblotted onto a PVDF membrane in a 50mM Tris-Base, 20% methanol, 40 mM glycine electrophoresis buffer. Membranes were incubated in 5% nonfat dry milk in

TBST (60mM Tris-Base, 120mM NaCl, 0.2% Tween 20) for 1 hour. Blots were probed

with primary antibody overnight at 4°C in 2% BSA in TBST, and then incubated with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling) in 5% dry milk in

TBST for 1 hour at room temperature. Bound antibodies were detected by chemiluminescence with ECL detection reagents (Amersham Pharmacia Biotech) and visualized by autoradiography. The primary antibodies used for Western blot analysis were: a mouse monoclonal anti-PDEF antibody (Gu et al., unpublished data), mouse anti-

Vimentin (Santa Cruz Biotechnology Inc. (Santa Cruz, CA)), mouse anti-fibronectin

(Santa Cruz Biotechnology Inc. (Santa Cruz, CA)) and mouse anti-cytokeratin18 (Santa

Cruz Biotechnology,Inc. (Santa Cruz, CA)). Mouse anti- (Sigma-Aldrich, (St.

Louis, MO)) was used as a control for loading. Experiments were repeated at least three- times, and representative data are shown.

Immunofluorescence

Cells cultured on glass coverslips were rinsed twice with PBS (pH 7.4), and fixed with

4% (w/v) formaldehyde in PBS for 20 min at room temperature. The fixed cells were

washed 3X10 min with PBS, and permeabilized with 0.3% Triton X-100 in PBS for 5

min. They were then incubated with PBS containing 1% BSA and 0.3% Triton X-100 for

1h at room temperature, which was followed by incubation with primary antibodies in

PBS containing 1% BSA and 0.3% Triton X-100 overnight at 40C. After washing with

PBS containing 1% BSA and 0.3% Triton X-100 buffer 3 X 10 min, the cells were

incubated with secondary antibodies (conjugated with fluorescent probes) in PBS with BSA and Triton X-100 for 30 min at room temperature. After washing 3 X 10 min, each

coverslip was mounted on a glass slide with the cell side down in a 1:1 solution of

PBS/glycerol. Fluorescent signals were observed with an Alexa Fluor 633-conjugated

goat anti-mouse antibody (1:500). Antibodies used were vimentin: mouse monoclonal

antibody (dilution 1:250) and 18: mouse monoclonal antibody (dilution

1:200). F- was stained with PHALLOIDIN (Tritc labeled, 1:1000 dilution, Sigma).

Northern Blot analysis

Total RNA was extracted using the Qiagen RNA mini Kit according to the instructions

from the manufactruer (Qiagen,). The amount of RNA sample was quantified by

spectrophotometry. RNA (20 ug) was loaded onto a 1.5% formaldehyde agarose gel and

subjected to electrophoresis followed by transer to a nylon membrane (Boehringer

Mannheim, Indianapolis, IN, USA). The membrane was UV-crosslinked. Equal loading

of the samples was verfied by checking the expression of GAPDH. A 32P-dUTP labeled

PDEF cDNA probe was used to hybridize to the membrane under standard conditions.

The blots were exposed to Kodak XAR film (Fisher) at room temperature.

Microarray analysis

Total RNA was harvested from two clones each of PC-3 cells stably expressing PDEF

siRNA or GFP siRNA using QIAshredder (Qiagen) and RNeasy Mini Kit (Qiagen). 1 ug

of total RNA from each sample were used to robotically prepare antisense biotinylated

RNA on the Affymetrix GeneChip Array Station (Affymetrix, Santa Clara, CA, USA).

Synthesis of single-stranded cDNA was done with the use of T7-oligo (dT) primer using the GeneChip HT One-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA, USA).

The GeneChip HT IVT Labeling Kit was used to convert cDNA into cRNA. The

biotinylated cRNAs were subsequently fragmented and mixed with hybridization

cocktails, and .hybridized onto the Affymetrix HT-U133AA of Av2 GeneChip (a high

throughput platform array in a 24-well format, compatible to HG-U133A chip, and

currently unavailable in the market) at 480C over 16 h. Array washing and staining were done using the Affymetrix GeneChip Array Station (Caliper Life Science, Mountain

View, CA, USA) following a robotic protocol according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA, USA). Arrays were then scanned by the

GeneChip HT Scanner (Affymetrix), an automated epi-fluorescence imaging scanner, and signal intensity for each transcript (background-subtracted and adjusted for noise) was determined using Genechip operating software 2.0. The scanned array images were analyzed by dChip (7). A recent study has suggested that regarding signal value calculation dChip is more robust than Affymetrix software Microarray Analysis Suite

(MAS) 5.0 for about 60% of the genes (8). In the dChip analysis a smoothing spline

normalization method was applied prior to obtaining model-based gene expression

indices, a.k.a. signal values. There were no outliers identified by dChip so all samples

were carried on for subsequent analysis.

When comparing two groups of samples to identify genes enriched in a given group, we

used the lower confidence bound (LCB) of the fold change (FC) between the two groups

as the cut-off criteria. If 90% LCB of FC between the two groups was above 1.2, the

corresponding gene was considered to be differentially expressed. LCB is a stringent

estimate of the FC and has been shown to be the better ranking statistic (9). Recently, dChip’s LCB method for assessing differentially expressed genes have been shown to be

superior to other commonly used approaches, such as MAS 5.0 and Robust Multiarray

Average (RMA) (10) based methods (11)

By use of LCB, we can be 90% confident that the actual FC is some value above the

reported LCB. It was suggested by a study exploring the accuracy and calibration of

Affymetrix chips using custom arrays and quantitative reverse transcriptase real-time

PCR (QRTPCR) assays that the chip analyses underestimate differences in gene expression (12). In regard to their work, and by (13), a criterion of selecting genes that

have a LCB above 1.2 most likely corresponds to genes with an “actual” fold change of at least 3 in gene expression.

Legends:

Supplemental Table 1. Target genes for PDEF in integrin, VEGF, Wnt, TGF-β

pathways.

Supplemental Table 2. The selected list of gene enrichment clusters (Score >2)

obtained using DAVID database and integrated tools (14).

Supplemental Figure 1. PDEF depletion decreases the adhesion ability of PC-3 cells.

PC-3 cells were infected with Lentiviruses encoding PDEF siRNA or control GFP siRNA

and plated on the top of gel. 3 hours after plating, cells were fixed, stained and

photographed. Representative images of adherent cells were shown.

Supplemental Figure 2. Down regulation of PDEF results in increased PC-3 cell

migration and invasion.

A, PC-3 cells with siRNA for GFP (control) or PDEF (knock down PDEF) were cultured

on 24-well transwell plates coated with Fibronectin. 16 h later, the cells that migrated

through the pores of the membrane to the other side were fixed, stained and

photographed. B, Invasion Assay in transwell plates coated with Matrigel after 24 h

incubation and 10% serum as chemoattractant. The invaded cells were fixed, stained and

photographed. Representative images of migrated and invaded cells are shown.

Supplemental Figure 3. Pathway analysis of PDEF regulated genes in PC-3 cells.

The list of genes up- and down-regulated in PC-3 cells expressing the PDEF siRNA was

imported into the Ingenuity Pathway analysis tool and canonical pathways were

determined. The Integrin, TGF-β, VEGF, Wnt pathways are shown here (Supplemental

Figure 3). Red colour symbolizes genes that are upregulated in PDEF siRNA expressing cells relative to the control cells and green colour represents genes that are downregulated. Blank symbols represent genes whose expression did not change significantly.

Supplemental Figure 4. Colorgram of highly enriched (Score 8.07) signaling

pathways related to differentially expressed genes derived using the DAVID

database (14). The top part of the colorgram is shown in four panels. The green color indicates the genes positively linked to a biological process, whereas black color indicates no information is available about gene relation to a biological process or term. The panels depict the genes of pathways (Wnt, Notch, cadherin, TGF-β, etc) that are

important for cell migration, invasion, and adhesion.

Supplemental Figure 5. Colorgram of pathways that are enriched among the

differentially expressed genes. The green color indicates the genes are positively linked

to a pathway, whereas black color indicates no information is available about gene

relation to a pathway. A) Notch signaling and related pathways, B) Wnt pathways, C)

TGF-β and related pathways and D) Integrin and related pathways.

Supplemental Figure 6. Northern blot analysis for the down-regulation of PDEF by

TGF-β in prostate cancer cells.

Northern blot analysis of PDEF expression in PC-3 cells incubated in the absence or presence of TGF-β for 48h. Northern blots were probed with 32P-dUTP-labeled PDEF

cDNA. The blots were exposed to Kodak XAR film at room temperature. GAPDH probe

was used to show equal loading.

Supplemental Figure 7. Potential binding sites for PDEF, or Smad in the human

Snail2 promoter. Underlined red nucleotides indicate the potential binding sites.

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Supplemental Table 1

Up-regulated genes by siPDEF

Gene Title Gene Symbol NP-siGF NP-siGFP NP-siPDE NP-siPDEFFC LCB cadherin 11, type 2, OB-cadherin (osteoblast) CDH11 1014.8 998.45 2289.07 2320 2.29 2.12 cadherin 11, type 2, OB-cadherin (osteoblast) CDH11 285.83 244.59 600.52 657.83 2.34 1.92 cadherin 18, type 2 CDH18 7.43 3.06 122.16 124.84 21.4 2.53 cadherin 2, type 1, N-cadherin (neuronal) CDH2 373.69 354.04 595.29 623.78 1.67 1.48 cadherin 2, type 1, N-cadherin (neuronal) CDH2 927.47 897.65 1355.31 1435.26 1.54 1.39 collagen, type I, alpha 1 COL1A1 151.07 146.63 436.9 538.77 3.27 2.32 collagen, type IV, alpha 1 COL4A1 171.17 169.29 1284.25 1089.01 6.98 5.59 collagen, type IV, alpha 1 COL4A1 242.08 207.67 1423.73 1238.71 5.87 4.73 collagen, type IV, alpha 2 COL4A2 119.84 110.83 533.58 480.8 4.4 3.57 collagen, type IV, alpha 2 COL4A2 392.27 371.69 1836.7 1395.6 4.2 3.1 collagen, type IV, alpha 5 (Alport syndrome) COL4A5 252.1 250.88 662.38 623.69 2.55 2.17 collagen, type IV, alpha 6 COL4A6 106.8 80.31 179.25 196.9 2.02 1.53 collagen, type V, alpha 1 COL5A1 46.91 52.6 224.18 237.27 4.7 3.43 collagen, type V, alpha 1 COL5A1 146.9 143.9 464.41 338.8 2.77 1.71 collagen, type V, alpha 1 COL5A1 55.72 49.33 245.57 134.28 3.56 1.57 collagen, type V, alpha 2 COL5A2 163.33 158.25 338.87 376.19 2.22 1.89 collagen, type V, alpha 2 COL5A2 141.63 157.36 275.25 338.82 2.08 1.61 collagen, type VI, alpha 1 COL6A1 544.51 447.49 1017.25 1194.18 2.23 1.79 collagen, type VI, alpha 1 COL6A1 4107.8 4019.02 6989.55 6960.97 1.72 1.59 collagen, type VI, alpha 1 COL6A1 845.01 699.29 1192.03 1757.57 1.91 1.27 collagen, type VI, alpha 1 COL6A1 347.01 302.06 478.4 658.61 1.76 1.25 collagen, type VI, alpha 2 COL6A2 1162.7 1012.16 3697.75 3406.79 3.27 2.79 collagen, type VI, alpha 2 COL6A2 248.64 261.26 501.17 431.28 1.83 1.49 collagen, type VI, alpha 3 COL6A3 477.51 481.47 4412.46 3833.38 8.6 7.19 collagen, type XIII, alpha 1 COL13A1 963.59 847.65 2256.4 2398.36 2.58 2.27 collagen, type XIII, alpha 1 COL13A1 347.53 279.27 811.25 932.24 2.8 2.22 collagen, type XIII, alpha 1 COL13A1 216.84 176.47 553.78 532.14 2.8 2.19 collagen, type XVI, alpha 1 COL16A1 42.64 57.79 105.99 123.86 2.41 1.54 connective tissue growth factor CTGF 296.49 402.69 778.7 908.39 2.41 1.84 integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor) ITGA3 924.37 746.22 1705.12 1504.59 1.92 1.57 integrin, alpha 5 (fibronectin receptor, alpha polypeptide) ITGA5 176.15 140.51 308.08 292.27 1.91 1.5 integrin, alpha 6 ITGA6 2064.9 2123.31 2623.05 2642.27 1.26 1.21 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 SERPINA1 425.1 409.03 1376.85 1603.19 3.57 3.03 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 SERPINA1 558.85 523.08 1458.94 1405.59 2.66 2.25 serpin peptidase inhibitor, clade B (ovalbumin), member 3 SERPINB3 10.63 29.78 74.87 62.42 3.42 1.3 serpin peptidase inhibitor, clade B (ovalbumin), member 7 SERPINB7 1617.5 1808.41 2538.56 2524.38 1.46 1.3 serpin peptidase inhibitor, clade D (heparin cofactor), member 1 SERPIND1 61.67 73.27 866.48 990.03 13.5 9.5 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 SERPINE1 618.57 587.87 3754.35 3888.27 6.34 5.56 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 SERPINE1 1383.4 1644.88 7150.48 7415.46 4.82 3.85 serpin peptidase inhibitor, clade I (neuroserpin), member 1 SERPINI1 22.06 24.24 69.17 49.91 2.65 1.34 SMAD, mothers against DPP homolog 1 (Drosophila) SMAD1 63.14 55.35 109.88 108.43 1.82 1.32 SMAD, mothers against DPP homolog 2 (Drosophila) SMAD2 419.99 410.66 644.97 639.79 1.54 1.38 SMAD, mothers against DPP homolog 2 (Drosophila) SMAD2 355.57 356.59 535.51 510.92 1.47 1.34 SMAD, mothers against DPP homolog 3 (Drosophila) SMAD3 1897.3 1848.82 2677.9 3004.8 1.51 1.35 SMAD, mothers against DPP homolog 3 (Drosophila) SMAD3 572.2 547.88 792.33 831.22 1.45 1.33 SMAD, mothers against DPP homolog 4 (Drosophila) SMAD4 256.71 244.22 461.2 433.75 1.79 1.57 snail homolog 2 (Drosophila) SNAI2 456.47 455.36 1038.47 1037.91 2.28 2.03 syndecan 1 SDC1 542.32 633.83 1300.65 1325.14 2.25 1.96 syndecan 1 SDC1 1135.4 1013.9 1915 1765.27 1.74 1.5 syndecan 2 (heparan sulfate proteoglycan 1, cell surface-associated, fibroglycan) SDC2 64.36 65.42 367.7 332.57 5.4 3.73 syndecan 2 (heparan sulfate proteoglycan 1, cell surface-associated, fibroglycan) SDC2 211.28 237.56 490.15 439.49 2.07 1.63 tenascin C (hexabrachion) TNC 1200.5 1201.14 2319.85 2027.3 1.81 1.59 transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47) TCF3 288.45 284.77 442.58 397.88 1.46 1.25 Transcription factor 4 TCF4 112.07 104.62 265.48 209.5 2.22 1.61 Transcription factor 4 TCF4 133.56 172.13 324.38 278.51 1.96 1.48 transcription factor 4 TCF4 112.61 109.31 161.54 169.03 1.49 1.23 transcription factor 8 (represses interleukin 2 expression) TCF8 20.79 2.5 285.6 159.38 20.7 4.92 transcription factor 8 (represses interleukin 2 expression) TCF8 49.78 3.23 297.86 200.95 9.27 3.16 transforming growth factor, beta 1 (Camurati-Engelmann disease) TGFB1 357.27 351.25 488.32 493.63 1.39 1.23 transforming growth factor, beta-induced, 68kDa TGFBI 1314.2 1240.57 6670.38 6554.49 5.19 4.74 vimentin VIM 2177.8 2368.09 8418.08 8901.66 3.82 3.38 , beta 2 (laminin S) LAMB2 178.01 157.79 333.95 271.03 1.8 1.39 laminin, gamma 1 (formerly LAMB2) LAMC1 386.1 457.55 1020.75 814.37 2.16 1.66 laminin, gamma 1 (formerly LAMB2) LAMC1 466.78 463 753.11 663.5 1.52 1.34 laminin, gamma 2 LAMC2 2151.6 2060.77 3333.09 2937.03 1.48 1.31 vascular endothelial growth factor VEGF 642.24 594.44 1121.85 1294.89 1.96 1.67 vascular endothelial growth factor VEGF 2249 2205.51 3146.71 3854.32 1.57 1.3 vascular endothelial growth factor VEGF 464.01 442.23 671.04 1029.51 1.88 1.21 vascular endothelial growth factor VEGF 640.16 670.35 934.18 1376.03 1.76 1.2 vascular endothelial growth factor B VEGFB 85.33 114.77 191.64 178.67 1.85 1.33 hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor) HIF1A 4700.1 5205.05 7040.9 7841.05 1.49 1.3 frizzled homolog 1 (Drosophila) FZD1 133.51 140.38 185.9 210.98 1.46 1.21 frizzled homolog 4 (Drosophila) FZD4 104.71 105.2 213.26 217.89 2.05 1.5 frizzled homolog 8 (Drosophila) FZD8 154.58 94.98 549.11 580.78 4.47 3.08 lymphoid enhancer-binding factor 1 LEF1 17.93 14.33 215.9 158.58 11.8 4.02 lymphoid enhancer-binding factor 1 LEF1 7.68 12.31 88.4 60.81 7.35 2.38 dickkopf homolog 3 (Xenopus laevis) DKK3 1.25 0.92 57.48 41.07 43.3 2.08 wingless-type MMTV integration site family, member 5A WNT5A 256.83 232 559.27 569.59 2.29 1.99 wingless-type MMTV integration site family, member 5A /// wingless-type MMTV integration site family, member 5A WNT5A 78.08 89.24 251.94 199.66 2.72 1.95 phosphoinositide-3-kinase, class 3 PIK3C3 373.11 392.76 619.94 605.65 1.61 1.38 phosphoinositide-3-kinase, catalytic, delta polypeptide PIK3CD 79.5 46.88 131.33 194.41 2.61 1.4 phosphoinositide-3-kinase, catalytic, delta polypeptide /// phosphoinositide-3-kinase, catalytic, delta polypeptide PIK3CD 223.78 227.56 962.6 996.7 4.34 3.83 ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2) RAC2 563.31 567.98 808.65 835.6 1.45 1.33 ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2) RAC2 1389.4 1352.78 1826.98 1956.69 1.38 1.25 ras-related C3 botulinum toxin substrate 3 (rho family, small GTP binding protein Rac3) RAC3 65.94 29.03 166.29 111.14 2.94 1.37 Wiskott-Aldrich syndrome protein interacting protein WASPIP 36.83 61.3 159.37 159.19 3.23 2.03 dedicator of cytokinesis 1 DOCK1 140.42 136.66 352.7 292.23 2.33 1.82 dedicator of cytokinesis 10 DOCK10 152.97 158.09 274.62 263.14 1.72 1.43 dedicator of cytokinesis 4 DOCK4 294.51 304.49 707.39 643.54 2.27 1.94 phospholipase C, beta 4 PLCB4 144.62 142.11 394.46 382.46 2.71 2.28 phospholipase C, beta 4 PLCB4 214.03 236.77 426.25 510.45 2.08 1.68 Phospholipase C, epsilon 1 PLCE1 72.99 92.85 143.51 134.75 1.66 1.27 phospholipase C, gamma 1 PLCG1 265.48 229.88 445.39 450.85 1.8 1.52 fibronectin 1 FN1 388.65 394.31 734.12 841.16 2.01 1.71 protein kinase C, alpha PRKCA 170.62 188.61 1183.49 1159.5 6.53 5.68 PTK2 protein tyrosine kinase 2 PTK2 1174.9 1062.14 2140.48 1946.12 1.82 1.6 PTK2 protein tyrosine kinase 2 PTK2 2353.8 2430.95 3554.75 3636.95 1.51 1.43 mitogen-activated protein kinase kinase kinase kinase 4 MAP4K4 777.24 656.99 1278.05 1276.64 1.79 1.53 mitogen-activated protein kinase kinase kinase kinase 4 MAP4K4 1207.6 1275.72 1735.99 1860.5 1.45 1.32 mitogen-activated protein kinase 9 MAPK9 770.6 787.78 1068.64 1119.17 1.4 1.3 mitogen-activated protein kinase-activated protein kinase 2 MAPKAPK2 201.5 210.78 340.23 342.93 1.65 1.44 mitogen activated protein kinase binding protein 1 MAPKBP1 379.46 388.61 543.27 547.19 1.42 1.31 histone deacetylase 1 HDAC1 3753.1 3578.75 5065.16 5088.5 1.39 1.3 bone morphogenetic protein 2 BMP2 25.47 -3.97 92.34 56.37 6.98 1.56

Down-regulated genes by siPDEF cytokeratin type II K6HF 5099.7 5016.18 1924.71 1949.92 -2.61 -2.4 cadherin 1, type 1, E-cadherin (epithelial) CDH1 785.17 719.82 296.45 287.88 -2.58 -2.3 cadherin 1, type 1, E-cadherin (epithelial) CDH1 3050.7 3135.21 1646.33 1401.41 -2.03 -1.8 cadherin 20, type 2 CDH20 61.01 79.24 -120.13 -159.11 -70.9 -1.8 cadherin 3, type 1, P-cadherin (placental) CDH3 2161.7 1890.06 608.15 685.68 -3.15 -2.6 18 KRT18 9115 9315.99 4830.58 4961.83 -1.89 -1.8 KRT19 6753.3 6734.3 5121.84 5196.44 -1.31 -1.3 KRT3 58.05 45.89 9.12 -11.67 -52.4 -1.3 /// /// keratin 6C /// keratin 6E KRT6A/B/C/E 196.76 193.98 40.43 63.27 -3.65 -2.1 keratin 6A /// keratin 6C /// keratin 6E KRT6A/C/E 366.63 318.1 49.37 57.74 -6.39 -3.3 keratin 6B KRT6B 611.24 589.96 137.77 112.12 -4.79 -3.9 keratin 6B KRT6B 219.92 186.45 71.13 51.63 -3.21 -2 KRT7 4000.6 3966.27 2698.57 2629.59 -1.49 -1.4 /// keratin 8 KRT8 2222 2025.33 1191.68 1037.79 -1.92 -1.7 keratin associated protein 9-9 /// keratin associated protein 9-5 /// keratin associated protein 9-5 KRTAP9-9/9-5 45.43 22.15 6.2 6.44 -5.46 -1.3 keratin, , acidic, 3A KRTHA3A 197.46 253.28 76.6 71.58 -3.05 -2.1 keratin, hair, acidic, 4 KRTHA4 1058.6 1121.4 403.01 368.92 -2.83 -2.4 integrin, beta 4 ITGB4 592.86 577.62 370.73 322.09 -1.69 -1.4 integrin, beta 4 ITGB4 243.37 205.24 97.58 131.99 -1.95 -1.4 integrin, beta 6 ITGB6 168.07 197.07 112.93 64.29 -2.09 -1.3 integrin, beta 6 ITGB6 172.43 195.71 117.38 77.82 -1.9 -1.3 integrin, beta 8 ITGB8 100.76 137.98 14.67 29.96 -5.21 -2.7 integrin, beta 8 ITGB8 149.27 167.51 51.27 79.51 -2.43 -1.6 phosphoinositide-3-kinase, catalytic, beta polypeptide PIK3CB 194.02 192.98 106.62 117.41 -1.72 -1.4 phosphoinositide-3-kinase, regulatory subunit 3 (p55, gamma) PIK3R3 156.31 150.62 77.31 80.44 -1.95 -1.6 Phosphoinositide-3-kinase, regulatory subunit 4, p150 PIK3R4 21.76 53.27 -2.86 6.25 -27 -1.3 phosphatidylinositol 4-kinase, catalytic, beta polypeptide PIK4CB 341.03 376.83 242.02 197.58 -1.64 -1.3 phosphatidylinositol-4-phosphate 5-kinase, type II, gamma PIP5K2C 1713.1 1779.97 1034.94 1017.51 -1.7 -1.6 parvin, alpha PARVA 938.5 983.08 588.59 529.22 -1.73 -1.5 transcription factor 1, hepatic; LF-B1, hepatic nuclear factor (HNF1), albumin proximal factor TCF1 80.64 85.81 41.03 22.24 -2.73 -1.5 Transforming growth factor, beta 2 TGFB2 669.37 766.84 237.98 302.83 -2.64 -2.1 transforming growth factor, beta 2 TGFB2 2014.1 1965.84 1000.15 1033.68 -1.96 -1.9 transforming growth factor, beta 2 TGFB2 1521.9 1584.62 739.77 813.03 -2.01 -1.8 Thrombospondin 1 THBS1 522.74 557.55 105.58 81.71 -5.82 -4.2 thrombospondin 1 THBS1 3231.3 3687.56 962.69 1105.33 -3.35 -2.8 thrombospondin 1 THBS1 7176 7839.73 3799.49 3515.42 -2.05 -1.8 thrombospondin 1 THBS1 6755 6779.1 4120.91 4020.33 -1.66 -1.6 thrombospondin 1 THBS1 6654.1 6408.76 4467.46 4207.1 -1.5 -1.4 frizzled homolog 10 (Drosophila) FZD10 144.85 123.6 50.44 77.75 -1.96 -1.2 frizzled homolog 5 (Drosophila) FZD5 51.28 43.49 -18.77 4.17 -46.7 -1.5 eukaryotic translation initiation factor 1A, X-linked EIF1AX 3014.6 3150.14 2342.82 2442.69 -1.29 -1.2 eukaryotic translation initiation factor 2B, subunit 2 beta, 39kDa EIF2B2 1757.3 1833.78 1288.33 1401.86 -1.35 -1.2 eukaryotic translation initiation factor 3, subunit 8, 110kDa EIF3S8 3609 3470.99 2488.18 2577.89 -1.4 -1.3 eukaryotic translation initiation factor 3, subunit 8, 110kDa EIF3S8 5739.3 5801.2 4634.02 4560.82 -1.26 -1.2 eukaryotic translation initiation factor 3, subunit 8, 110kDa EIF3S8 4482.8 4520.98 3509.51 3594.48 -1.27 -1.2 forkhead box A1 FOXA1 790.99 747.08 468.7 443.79 -1.67 -1.5 Forkhead box C1 FOXC1 141.62 172.62 86.61 96.38 -1.7 -1.3 forkhead box O1A (rhabdomyosarcoma) FOXO1A 186.08 195.59 71.52 76.02 -2.58 -2.1 forkhead box O3A FOXO3A 390.48 414.16 249.05 299.62 -1.47 -1.2 vascular endothelial growth factor C VEGFC 2404.9 2108.01 1559.11 1478.47 -1.48 -1.3 inhibin, beta B (activin AB beta polypeptide) INHBB 2885.9 2516.41 1541.33 1557.8 -1.74 -1.5 NK2 transcription factor related, locus 5 (Drosophila) NKX2-5 79.17 57.84 8.37 -10.49 -69.2 -1.5 SMAD specific E3 ubiquitin protein ligase 1 SMURF1 123.28 158.27 75.48 65.93 -2.02 -1.4 Table S2: The selected list of gene enrichment clusters (Score >2) obtained using DAVID database and integrated tools.

Terms Genes Pvalue Enrichment Score :11.43 negative regulation of biological process 162 7.67E-14 negative regulation of cellular process 153 1.02E-13 negative regulation of cellular physiological process 133 8.80E-11 negative regulation of physiological process 135 2.81E-10 Enrichment Score :8.07 signal 384 3.07E-23 glycoprotein 469 3.04E-20 extracellular region 190 0.0025 signal peptide 379 0.004287 glycosylation site:N-linked (GlcNAc...) 429 0.169937 disulfide bond 320 0.232529 Enrichment Score :5.25 golgi stack 66 1.56E-07 signal-anchor 54 1.17E-06 Golgi apparatus 87 5.48E-05 Golgi stack 70 1.01E-04 Enrichment Score :5.1 cellular morphogenesis 65 6.61E-08 growth 46 6.16E-06 regulation of cell size 39 1.03E-05 cell growth 39 1.03E-05 regulation of cell growth 31 5.47E-05 regulation of growth 32 1.05E-04 Enrichment Score :5.1 glycoprotein 469 3.04E-20 membrane 504 4.82E-18 transmembrane 428 2.81E-06 plasma membrane 273 2.95E-06 intrinsic to plasma membrane 191 1.86E-04 integral to plasma membrane 189 2.52E-04 glycosylation site:N-linked (GlcNAc...) 429 0.169937 membrane 669 0.925312 transmembrane region 407 0.938666 intrinsic to membrane 496 0.981346 integral to membrane 494 0.98365 Enrichment Score :4.8 nuclear protein 445 1.11E-20 transcription 223 2.14E-12 transcription regulation 217 3.60E-10 regulation of biological process 548 4.03E-09 transcription regulator activity 220 7.14E-09 regulation of cellular process 514 1.30E-08 -binding 210 2.53E-08 regulation of cellular physiological process 479 1.14E-06 regulation of physiological process 491 1.46E-06 transcription factor activity 154 8.40E-06 DNA binding 281 0.00634 nucleus 524 0.032845 transcription, DNA-dependent 287 0.062838 regulation of metabolism 326 0.070899 regulation of transcription, DNA-dependent 277 0.084362 regulation of cellular metabolism 315 0.091752 nucleobase, nucleoside, nucleotide and nucleic acid metabolism 445 0.115144 transcription 306 0.125359 regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism 296 0.129471 regulation of transcription 291 0.142439 nucleic acid binding 400 0.213226 Enrichment Score :4.8 nuclear protein 445 1.11E-20 cellular physiological process 1381 2.25E-07 intracellular 1078 1.34E-04 membrane-bound organelle 774 2.58E-04 intracellular membrane-bound organelle 773 3.03E-04 organelle 890 0.007705 intracellular organelle 889 0.008418 nucleus 524 0.032845 cell 1506 0.886952 Enrichment Score :4.8 positive regulation of cellular process 106 1.23E-07 positive regulation of biological process 119 2.06E-07 positive regulation of physiological process 88 1.56E-05 positive regulation of cellular physiological process 85 1.75E-05 positive regulation of cellular metabolism 30 0.010091 positive regulation of metabolism 31 0.017785 Enrichment Score :4.3 apoptosis 47 3.17E-06 regulation of apoptosis 70 1.89E-05 regulation of programmed cell death 70 2.28E-05 negative regulation of apoptosis 35 4.42E-05 negative regulation of programmed cell death 35 5.14E-05 apoptosis 98 6.21E-05 programmed cell death 98 7.14E-05 death 100 1.37E-04 cell death 99 1.67E-04 anti-apoptosis 30 2.16E-04 Enrichment Score :3.9 transcription factor binding 64 7.58E-07 transcriptional activator activity 45 1.56E-04 transcription cofactor activity 49 1.62E-04 transcription coactivator activity 28 0.012063 Enrichment Score :3.77 cell migration 28 4.63E-05 localization of cell 48 2.61E-04 locomotion 48 2.61E-04 cell motility 48 2.61E-04 Enrichment Score :3.72 heparin-binding 19 1.42E-06 polysaccharide binding 27 4.37E-05 pattern binding 28 9.42E-05 heparin binding 21 1.86E-04 glycosaminoglycan binding 24 4.46E-04 carbohydrate binding 45 0.003838 heparin binding 9 0.004653 Enrichment Score :3.63 regulation of progression through cell cycle 86 3.12E-05 regulation of cell cycle 86 3.37E-05 cell cycle 115 5.01E-04 negative regulation of progression through cell cycle 31 0.005519 Enrichment Score :3.61 extracellular matrix 37 6.84E-05 extracellular matrix structural constituent 25 3.63E-04 extracellular matrix (sensu Metazoa) 60 3.84E-04 extracellular matrix 61 3.94E-04 Enrichment Score :3.59 organ morphogenesis 51 2.28E-06 angiogenesis 20 2.63E-04 blood vessel development 20 4.74E-04 vasculature development 20 4.74E-04 blood vessel morphogenesis 20 4.74E-04 angiogenesis 10 0.004384 Enrichment Score :3.39 negative regulation of metabolism 51 4.13E-05 negative regulation of cellular metabolism 45 5.80E-05 negative regulation of transcription 37 3.60E-04 negative regulation of nucleobase, nucleoside, nucleotide and nucleic acid metab 38 5.42E-04 negative regulation of transcription, DNA-dependent 22 0.025444 Enrichment Score :3.33 protein kinase inhibitor activity 13 3.83E-05 protein kinase inhibitor 8 1.05E-04 kinase inhibitor activity 13 1.52E-04 protein kinase regulator activity 15 0.003882 kinase regulator activity 16 0.009612 Enrichment Score :3.24 enzyme regulator activity 115 1.37E-06 enzyme activator activity 42 7.97E-04 GTPase regulator activity 48 0.003316 GTPase activator activity 25 0.029114 Enrichment Score :3.07 molecular function unknown 106 5.94E-05 biological process unknown 102 2.63E-04 cellular component unknown 98 0.039422 Enrichment Score :2.82 egf-like domain 48 2.84E-08 IPR013032:EGF-like region 67 5.42E-06 IPR006209:EGF-like 66 6.51E-06 IPR006210:EGF 47 1.03E-04 IPR000742:EGF-like, type 3 49 1.19E-04 IPR013091:EGF calcium-binding 21 4.16E-04 IPR000152:Aspartic acid and asparagine hydroxylation site 26 6.88E-04 domain:EGF-like 1 23 0.001106 IPR001881:EGF-like calcium-binding 28 0.002037 domain:EGF-like 11; calcium-binding 7 0.002139 domain:EGF-like 3 17 0.002208 SM00181:EGF 39 0.0072 domain:EGF-like 2 17 0.007459 domain:EGF-like 5; calcium-binding 10 0.011163 domain:EGF-like 2; calcium-binding 12 0.012854 domain:EGF-like 7; calcium-binding 8 0.017495 SM00179:EGF_CA 22 0.02319 domain:EGF-like 4 11 0.02797 domain:EGF-like 6; calcium-binding 8 0.028201 domain:EGF-like 3; calcium-binding 8 0.034926 domain:EGF-like 4; calcium-binding 8 0.042633 domain:EGF-like 1; atypical 3 0.083923 Enrichment Score :2.71 binding 1396 4.96E-10 cellular physiological process 1381 2.25E-07 cellular process 1581 0.00294 primary metabolism 942 0.052022 physiological process 1523 0.180532 metabolism 1023 0.259066 cellular metabolism 952 0.302495 cell 1506 0.886952 Enrichment Score :2.71 domain:Helix-loop-helix motif 24 1.68E-04 DNA-binding region:Basic motif 29 9.18E-04 IPR001092:Basic helix-loop-helix dimerisation region bHLH 25 0.00179 IPR011598:Helix-loop-helix DNA-binding 23 0.008018 SM00353:HLH 24 0.013362 Enrichment Score :2.7 cytoskeletal protein binding 68 5.90E-06 actin-binding 29 0.00316 actin binding 42 0.005083 actin 33 0.166047 Enrichment Score :2.62 cell organization and biogenesis 245 2.24E-07 protein transport 52 3.17E-04 establishment of cellular localization 97 0.00235 cellular localization 97 0.003028 intracellular transport 95 0.003345 protein localization 87 0.030125 intracellular protein transport 51 0.030628 establishment of protein localization 84 0.036456 protein transport 81 0.042384 Enrichment Score :2.62 growth factor binding 20 1.26E-05 growth factor binding 7 3.39E-04 IPR000867:Insulin-like growth factor-binding protein, IGFBP 9 5.45E-04 insulin-like growth factor binding 10 6.77E-04 IPR000716:Thyroglobulin type-1 8 0.001432 domain:IGFBP 9 0.001552 domain:Thyroglobulin type-1 7 0.002139 SM00121:IB 9 0.003015 SM00211:TY 7 0.010977 SF001969:insulin-like growth factor binding protein 4 0.024936 IPR009168:Insulin-like growth factor binding protein 4 0.033634 h_ghrelinPathway:Ghrelin:Regulation of Food Intake and Energy Homeostasis 5 0.167869 Enrichment Score :2.59 vacuole 32 5.40E-04 lytic vacuole 27 0.0037 lysosome 27 0.0037 lysosome 16 0.005767 Enrichment Score :2.52 IPR001007:von Willebrand factor, type C 13 7.31E-04 SM00214:VWC 12 0.003151 domain:VWFC 7 0.011722 Enrichment Score :2.32 wound healing 25 5.87E-04 regulation of body fluids 25 0.001582 hemostasis 23 0.001872 blood coagulation 22 0.001888 coagulation 22 0.002499 blood coagulation 9 0.039886 HSA04610:COMPLEMENT AND COAGULATION CASCADES 15 0.184328 Enrichment Score :2.25 regulation of apoptosis 70 1.89E-05 regulation of programmed cell death 70 2.28E-05 positive regulation of apoptosis 29 0.027816 positive regulation of programmed cell death 29 0.029849 induction of apoptosis by extracellular signals 8 0.073191 induction of programmed cell death 24 0.085413 induction of apoptosis 24 0.085413 Enrichment Score :2.2 transferase 200 2.79E-11 nucleotide-binding 207 8.43E-11 atp-binding 155 3.43E-07 kinase 112 7.37E-05 nucleotide binding 277 9.34E-04 serine/threonine-protein kinase 53 0.002701 phosphotransferase 41 0.002995 purine nucleotide binding 237 0.003082 adenyl nucleotide binding 189 0.005588 ATP binding 182 0.007187 transferase activity 247 0.007553 phosphotransferase activity, alcohol group as acceptor 107 0.016379 IPR000719:Protein kinase 78 0.020566 active site:Proton acceptor 83 0.021364 IPR002290:Serine/threonine protein kinase 71 0.02467 nucleotide phosphate-binding region:ATP 116 0.025097 protein serine/threonine kinase activity 62 0.03503 domain:Protein kinase 64 0.045635 protein kinase activity 88 0.046153 IPR001245:Tyrosine protein kinase 66 0.054487 binding site:ATP 67 0.056626 kinase activity 126 0.066933 IPR008271:Serine/threonine protein kinase, active site 50 0.093927 protein-tyrosine kinase activity 33 0.099198 transferase activity, transferring phosphorus-containing groups 141 0.130139 protein phosphorylation 85 0.151721 protein kinase CK2 activity 27 0.180642 cAMP-dependent protein kinase activity 27 0.208069 cyclic nucleotide-dependent protein kinase activity 27 0.208069 SM00220:S_TKc 40 0.210664 phosphate metabolism 120 0.268431 phosphorus metabolism 120 0.268431 phosphorylation 94 0.524281 Enrichment Score :2.19 ATP-dependent RNA helicase activity 10 9.40E-04 helicase 25 0.001004 IPR001650:Helicase, C-terminal 25 0.001228 RNA-dependent ATPase activity 10 0.001279 IPR011545:DEAD/DEAH box helicase, N-terminal 24 0.001478 RNA helicase activity 11 0.003255 helicase activity 28 0.008756 SM00487:DEXDc 23 0.013581 SM00490:HELICc 22 0.028119 ATP-dependent helicase activity 19 0.05725 IPR001410:DEAD/DEAH box helicase 15 0.060127 IPR000629:ATP-dependent helicase, DEAD-box 8 0.065702 Enrichment Score :2.08 P-loop 35 1.14E-05 nucleotide binding 33 2.64E-05 gtp-binding 45 3.03E-04 prenylation 26 5.37E-04 GTP binding 19 0.001085 nucleotide phosphate-binding region:GTP 44 0.003127 GTPase activity 31 0.006126 IPR003578:Ras small GTPase, Rho type 23 0.011876 IPR005225:Small GTP-binding protein domain 29 0.014177 IPR003577:Ras small GTPase, Ras type 23 0.023959 IPR003579:Ras small GTPase, Rab type 25 0.03815 IPR001806:Ras GTPase 28 0.046905 IPR002041:GTP-binding nuclear protein Ran 15 0.055107 GTP binding 55 0.065995 lipid moiety-binding region:S-geranylgeranyl cysteine 18 0.080511 small GTPase mediated signal transduction 42 0.084522 guanyl nucleotide binding 55 0.085705 SM00175:RAB 12 0.136348 SF001710:Rab protein 14 0.154531 Enrichment Score :2.06 endocytosis 18 6.08E-04 coated pits 9 0.014697 coated pit 10 0.073693 Fig. S1

PC-3 siGFP PC-3 siPDEF Fig. S2A

siGFP; 1d (X200) siPDEF; 1d (X200)

Fig. S2B

PC-3 siGFP PC-3 siPDEF Fig. S3A Fig. S3B Fig. S3C Fig. S3D Fig. S4 Fig. S5 Fig. S6

PC-3

TGF-β1 0 7 ng/ul PDEF 2.0kb

GAPDH 1.3kb Fig. S7 GACTGGGTTC TTAAAACTTT AATCAATCTA TATTCATAAT AATATCCTTC AATTTCTTTT TTAGCATTAT ACAGGAAACT GGTAGATACT GAGATGGATT TTAATGGCTT TATACTGAGA AAATAGCACC ACATAAAAGC AGGGGAATAT TAGAAATAAA AATAATTGTC TCTAAAGACC CATACAACCC TTTTTCCCAT AAAAAAAAAG ATGCACTGTA ATACATGAAA AGATAAGATC TCTTGTCAAA AGTGTGAGAG AATGTCCGGT GGTTCCAAAT GACAGTTACC TCTTGCCCCC CTTCTCTGCC AGAGTTCCTT TTTATCTTTG CAATCTTCCA GTTGTTCCGA TCAGCCTGCC TTTAGAGGGC TACAAAGCAT TTCTTTCAAG CCACCATAGC TAACACGGTG ACATGAGTAC TTAATTTGCA CGCGGCCGCG CTGCCCCTGG CTTCGCGGAA GCCCTGAGTA GCGCAGCGCC CTCGCCGCAC GCAAGGCTGC AGTCCCGCTC CAGGCCAGAG TCCCAGGAGA GCGTCCTCCG CGCTCACAGG CGCCTTTGTC TTCCCGCTTC CCCCTTCCTT TTTCAAAAGC CAAGAGGTAA TTATTTGGTC TTTGTGCAAG GCAAACCTCT CCAGATGCCA CTTCCAAATA TAGGCTCTCA TTAACACCAG AGGCTGGCCT GGTGTGGTGC AGGGCGGCCC TTCCTTCTCC TGGCGGACAC TGTGTCCCCG CGCGCTGGCG CTGCACCACA TCTGGAAGCC AGGCGGGCAG GGCAGAGACC CCGGCTCCTG CGCCCCTCCT AGCTCCCAGA GAGCGTGGAT CGCGGGCGGG GCTCACCGAG CGAGGTTACC TCTCTTGAAA ATACTTAAAC ACTTTTTTTC CTCTCCACTG AAATCTCAAA AAACAGCCCA TTTTGAACCA GAATAATTTA GTCTGACAAC AGATTCTTCC TCTGTTCACA GCTGTCCCAG AGGGAGGAGC TGAAATCTGA ACCTCTCAGC TGTGATTGGA TCTTTCTTGC AAAAGAGAGG AAAAAAAAAC CCTCCCAGCC AAAACGGGCT CAGTTCGTAA AGGAGCCGGG TGACTTCAGA GGCGCCGGCC CGTCCGTCTG CCGCACCTGA GCACGGCCCC TGCCCGAGCC TGGCCCGCCG CGATGCTGTA GGGACCGCCG TGTCCTCCCG CCGGACCGTT ATCCGCGCCG GGCGCCCGCC AGACCCGCTG

Potential PDEF Binding Site: GGAA/T, A/TTCC Potential Smad Binding Site: AGAC, GTCT