Supporting InformationAslam et al. 10.1073/pnas.1403608111SI MethodsCell Culture.Primarymouse tumor cell cultureswere establishedandcultured as previously described (1). The mouse myoblast cell lineC2C12 was purchased from theAmerican TypeCulture Collection.Human alveolar rhabdomyosarcoma (aRMS) cell lines Rh5, Rh30,Rh3, and the embryonal rhabdomyosarcoma cell line Rh18 weregenerously provided by Peter Houghton of St. Jude Children’sResearch Hospital, Memphis, TN, the institution that originatedthe lines. Human cells were grown in RPMI 1640 supplementedwith 10% FBS and penicillin (100 U/mL)/streptomycin (100 μg/mL)(Invitrogen) in 5% CO2 in air at 37 °C.

Quantitative RT-PCR Analysis. Total RNA was isolated from mousetumors using TRIzol (Invitrogen) according to themanufacturer’sprotocol. RNA was subsequently purified using the RNeasyminiprep kit (Qiagen) per the manufacturer’s protocol, includingDNase treatment. SuperScript VILO cDNA Synthesis Kit wasused according to the manufacturer’s recommendations to gen-erate cDNA. Quantitative RT-PCR (RT-qPCR) was performedusing Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen)on an MJ Research DNA Engine Opticon 2 System following themanufacturer’s instructions. The level of mRNA expression foreach gene was normalized to β-glucuronidase (GusB). Primersequences for each gene were designed and optimized for ∼100%efficiency and the comparative Ct method. Invitrogen TaqManprimer probe was used for human PAX3:FOXO1 (catalog no.4351372). The following primers were used: Human ephrin B4(EPHB4): 5′-GAAGAAGGAGAGC TGTGTGGCAATC-3′ and5′-GATGACTGTGAACTGTCCGTCGTT-3′. Human EPHRINB2:5′-GCATCTGTCTGCTTGGT CTTTATCAAC-3′ and 5′-ATGG-CTGTGAGAAGGGACTCC-3′. Human platelet derived growthfactor β (PDGFRβ): 5′-CTCT CCATAGAAGCAATACTCTGT-GATGATG-3′ and 5′-GCAGAAGAAGCCACGTTACGAG-3′.HumanGUSB: 5′ CAGAAGCCCATTATT CAGAGCGAGT-3′and 5′-GCACTCTCGTCGGTGACTGT-3′. Human PAX3:FOXO1:TaqMan (Invitrogen, # 4351372). Mouse EphB4: 5′-TCT TCTAT-TACGAGAGCGAAGCTGA-3′ and 5′-CGCAGCGTCTTGAT-ATTAACTTTCCC-3′. Mouse EphrinB2: 5′ GGACTCTGTGTG-GAAGTACTGTTGG-3′ and 5′-TCTGTCTGCTTGGT CTTTA-TCAACC-3′. Mouse Pdgfrß: 5′-ATAACAGAAGACAGCGAGG-TGGACT-3′ and 5′-GTGCTGGAGATGTTGAGAACAAACT-3′.Mouse GusB: 5′-CAGAAGCCGATTATCCA GAGCGAGT-3′and5′-CAGCGGTGACTGGTTCGTCATG-3′.MousePax3:Foxo1:5′-AGA CAGCTTTGTGCCTCCAT-3′ and 5′-CTCTTGCCTCC-CTCTGGATT-3′.

Immunohistochemistry. Human aRMS tissue microarrays wereobtained from Cooperative Human Tissue Network as previouslymentioned (TMA #3000–30-P6897). Individual human aRMSsections were obtained from the Knight Cancer Institute TissueBank through institution-approved protocol. Histology was per-formed using standard protocol. Briefly, formalin-fixed paraffinembedded sections were dewaxed and dehydrated in xylene andgraded alcohol concentrations. Heat-induced epitope retrievalwas used with sodium citrate buffer (pH 6.0) for 20 min, slidesblocked with 2% (vol/vol) normal goat serum (NGS), and forendogenous biotin using an avidin/biotin blocking kit (VectorLabs). Primary antibodies for EphB4 (AP7625d, rabbit poly-clonal; Abgent), PDGFRβ (sc-339, rabbit polyclonal; Santa CruzBiotechnology), EphrinB2 (sc-1010, rabbit polyclonal; SantaCruz Biotechnology) were used at 1:50 overnight in 2% NGS.PDGF-BB (ab23914, rabbit polyclonal; Abcam) was used at

1:500 overnight in 2% NGS. Blocking peptides for each antibodywere purchased and used as follows: for EphB4 (BP7625d, Ab-gent) at 100 molar excess to antibody as recommended by themanufacturer, fivefold excess by weight to antibody for EphrinB2(sc-1010P) and PDGFRβ antibodies (sc-339P) from Santa Cruz.For PDGF-BB antibody, blocking peptide (ab24035; Abcam)was used at fivefold excess by weight to antibody. Primary anti-body was incubated in 2% NGS with or without blocking peptideovernight. VECTASTAIN Elite ABC kit (rabbit IgG) and Im-mPACT DAB Peroxidase Substrate kit was used according to themanufacturer’s protocol to visualize staining (Vector Lab). Slideswere counterstained with hematoxylin for 5 min, rinsed, dehy-drated, and mounted with xylene based mounting medium. Forthe tumor microarray, score was assigned on a scale from 1 to 3based on intensity of staining and percent cells deemed positive bya coauthor pathologist (A.M.). A score of 0 was given if <10% ofcells were stained positive, a score of 1 for >10% to <25%, a scoreof 2 for >25% to <50%, and a score of 3 for >50% or >25% to<50% with a relative strong intensity of stain. The pathologistdetermined that all tumor sections fell within these defined pa-rameters of staining intensity and percentage. Two sections werescored 0 for EphB4, 12 were scored 1, 8 were scored 2 and 9 werescored 3. One section was scored 0 for PDGFRβ, 8 were scored 1,9 were scored 2, and 13 were scored 3.

Immunoblotting and Immunoprecipitation. All cell lysates wereprepared using Cell Lysis Buffer (#9803, Cell Signaling Tech-nology) supplemented with protease and phosphatase inhibitors(Sigma) and subsequently standard immunoblotting procedureswere performed and proteins visualized with chemiluminescentsignal using Super Signal Western Pico or Dura Substrate(Pierce Biotechnology). Mouse tumors were washed in PBS thenlysed in RIPA buffer supplemented with protease and phos-phatase inhibitors. Lysates were homogenized and centrifuged atmaximum speed for 10 min at 4 °C, and supernatant used forWestern blotting. Antibodies used for immunoblotting were allobtained from Cell Signaling Technologies (used at 1:1,000) withthe exception of EphB4 antibody (1:500, sc-5536, rabbit poly-clonal; Santa Cruz Biotechnology), EphrinB2 (1:500, sc-15397,rabbit polyclonal; Santa Cruz Biotechnology), pan-Tyr antibody(1:10,000, 4G-10 mouse monoclonal; Upstate Biotechnology),t-Crkl (1:1,000, sc-319, rabbit polyclonal; Santa Cruz Biotechnology),and β-actin antibody (1:10,000, mouse monoclonal;Sigma). The p-Akt antibody from Cell Signaling recognizes thephosphorylated S473 residue of Akt (rabbit polyclonal, #9271).The p-FAK antibody from Cell Signaling recognizes the phos-phorylated Y576/577 residues of FAK (rabbit polyclonal, #3281).Immunoprecipitation was performed by standard protocol usingProtein A beads for rabbit IgG (Invitrogen) or Protein G beads formouse IgG (Millipore). For signaling studies involving siRNAknockdown, protein was extracted day 2 or day 3 posttransfection.For studies involving stimulation with PDGF-BB (ShenandoahBiotechnology) or mouse/human EphrinB2 (R&D Systems) in thepresence or absence of inhibitor, cells were serum-starved over-night and stimulated as indicated. Human IgG1 Fc (R&D Sys-tems) was used as a control for EphrinB2 clustered to humanIgG1 Fc. For EphB4 detection of C2C12 cells transiently trans-fected with Pax3:Foxo1 or control vector, Mouse EphB4 Antibodyfrom R&D Systems was used (#AF446).

RNA-Interference Studies. For all RNAi studies, pooled and in-dividual duplex siRNA were obtained from Dharmacon, including

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the mouse siRNA library targeting the tyrosine kinome (siGE-NOME). All experiments with siRNA are performed at 100 nMconcentration and include nonspecific pooled #1 siRNA as acontrol purchased from Dharmacon. For all experiments, unlessotherwise indicated, pooled siRNA targeting EphB4 or PDGFRβwas used. Transfection of siRNA was carried out using Lipofect-amine 2000 in Opti-MEM Reduced Serum Media (Invitrogen).Custom siRNA targeting human PAX3:FOXO1 was purchasedfrom Dharmacon, sequence of sense strand: 5′-CCUCUCACC-UCAGAAUUCATT-3′, antisense: 5′-UGAAUUCUGAGGUG-AGAGGTT-3′. Scrambled siRNA sequence for sense strand: 5′-CUACUAUACCGAUACUCCCTT-3′, antisense strand: 5′-GG-GAGUAUCGGUAUAGUAGTT-3′, as previously described (2).

Transient Transfection of C2C12 Cells.C2C12 cells were plated at 2 ×105 cells per well in a six-well plate. When cells were 80–90%confluent, 4 μg of plasmid DNA (pCDNA3 control or pCDNA3-Pax3:Foxo1), 10 μL Lipofectamine 2000 in Opti-MEM ReducedSerum Media was added to the cells per the manufacturer’sprotocol. After 48 h of incubation, the cells were harvested formRNA and protein expression.

In Vitro Growth Inhibition and Proliferation Assays. After cells wereplated in 96-well plates in the presence of inhibitor or siRNA, andincubated for 72 h or 96 h, respectively, 20-μL CellTiter 96AQueous One solution (MTS; Promega) was added to each welland absorbance values assessed by the BioTek Synergy 2 platereader (BioTek). For the PDGF-BB–stimulated cell proliferationassay, cells were plated in a 96-well plate in serum-free media andallowed to seed. Cells were then treated with indicated concen-trations of PDGF-BB ligand for 48 h, after which cell viability wasassessed by MTS assay for murine aRMS primary cell cultures and72 h for human aRMS cell lines.For experiments involving U23674 cells with PDGF-BB

stimulation alone or in the presence of imatinib and dasatinib,10,000 cells were plated per well in a 96-well plate in serum-freemedia and immediately thereafter serum-free media was addedwith combinations of vehicle alone, PDGF-BB (#GWB-9955E4;Genway Biotech) alone, or with imatinib or dasatinib. After a72-h incubation, cell viability was assessed using CellTiter-Gloluminescent cell viability assay (Promega) with a BioTek Synergy2 plate reader, as above.

Anchorage-Independent Colony Formation Assay. For soft agarassays, cells were transfected with nonspecific targeting siRNA, aswell as siRNA targeted to EphB4 or Pdgfrβ and on day 2, trypsi-nized and in each well of a six-well plate, 6,000 cells were sus-pended in 1.5 mL of 0.7% agarose (at 37 °C) in complete medium.The cells in agarose were plated atop a 1.4% agarose layer andwere allowed to grow for 2 wk before visualizing the colonies bylight microscopy.

Cell Viability and Apoptosis Assays. Cell viability was assessed byaddition of Guava ViaCount Reagent (Millipore) to cells andsubsequent analysis on the Guava Personal Cell Analysis System.For apoptosis assay analyzed by flow cytometry, cells were washedin a 2% FBS in PBS solution once, and subsequently resuspendedin 2% FBS in PBS solution. Equal amount Guava Nexin Reagentwas added to each sample and incubated at room temperatureprotected from light for 20 min. Cells were then analyzed ona Guava EasyCyte Flow Cytometer using CytoSoft 2.1.5 software.FCS files were exported and subsequently analyzed using FlowJoSoftware (Tree Star; PC v7.6).

In Vivo Studies. SCID/hairless/outbred (Crl:SHO-PrkdcscidHrhr)mice purchased from Charles River at 8 wk of age were injectedwith cardiotoxin in the right gastrocnemius muscle 1 d beforeinjection of 106 U23674 cells in the muscle. The length, width,and height of the tumors were measured with digital calipersdaily and the tumor volume was calculated from the formula:length × width × height × π/6. Once tumor volume reached 0.2 cm3

(2-wk postinjection), the tumor-bearing mice were treated withimatinib, dasatinib, or vehicle by oral gavage. Once tumor volumereached 2.0 cm3, mice were killed and the tumor was harvested atnecropsy. All animals were treated humanely and the experimentswere conducted in accordance with Oregon Health and ScienceUniversity Institutional Animal Care and Use Committee-approved protocols.

Statistical Analysis. Student t tests were done for determiningstatistical significance and a probability value of less than 0.05was accepted as significant. Wherever applicable, all of the ex-periments were done in triplicates and repeated twice unlessmentioned otherwise. Mantel–Cox analysis of survival curves wasdone using GraphPad Prism 5 software. For the microarray studyof Intergroup Rhabdomyosarcoma Study Group-IV samples (3),data were preprocessed with ProbeProfiler (Corimbia), whichmodels probe intensities across a data set to provide improvedsignal-to-noise ratio, identifies outliers, and adjusts for scannersaturation. To determine which genes were enriched amongsubsets of tumors, a Mann–Whitney–Wilcoxon test controlledfor a false-discovery rate < 0.05 was applied to compare thatsubset to the remaining tumors in the 89 nonduplicate tumordataset. The Mann–Whitney–Wilcoxon test controlled for afalse-discovery rate ≤ 0.05 was also applied to determine whichgenes were relatively unenriched in each subset of tumors.Failure-free survival analysis was performed by Cox regression,adjusting for known clinical covariates, which included histo-logical group, site, clinical group, surgical stage, age, and trans-location status.

1. Taniguchi E, et al. (2008) PDGFR-A is a therapeutic target in alveolar rhabdomyosarcoma.Oncogene 27(51):6550–6560.

2. Kikuchi K, et al. (2008) Effects of PAX3-FKHR on malignant phenotypes in alveolarrhabdomyosarcoma. Biochem Biophys Res Commun 365(3):568–574.

3. Blandford MC, et al. (2006) Rhabdomyosarcomas utilize developmental, myogenicgrowth factors for disease advantage: A report from the Children’s Oncology Group.Pediatr Blood Cancer 46(3):329–338.

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Fig. S1. RNA interference validation studies. (A) Average reduction in Pdgfrβ and EphB4 protein levels by immunoblot in U23674 and (B) U48484 cells. Datarepresents quantification of three individually performed immunoblots. (C) Murine aRMS U23674 and U48484 were transfected with indicated siRNA and 4 dlater the number of viable cells were determined. (D) U23674 cells were transfected with individual duplex siRNA which comprise the siGENOME pool and cellviability assessed by MTS assay. Concomitantly, RT-qPCR was done to determine if individual duplex siRNA resulting in decrease in cell viability correlated withdecrease in in mRNA levels of Pdgfrβ. (E) as well as for EphB4, confirming specificity of siRNA targeting. (F) U48484 cells were transfected with individualduplex siRNA which comprise the siGENOME pool and cell viability assessed by MTS assay. Concomitantly, RT-qPCR was done to determine if individual duplexsiRNA resulting in decrease in cell viability correlated with decrease in in mRNA levels of Pdgfrβ (G) as well as for EphB4, confirming specificity of siRNAtargeting. *P < 0.05.

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Fig. S2. RNA expression studies and Immunohistochemistry for EphB4 on a human aRMS tissue microarray. (A) RT-qPCR showing mRNA levels of EphB4 andEphrinB2 in both human aRMS and mouse aRMS compared with normal skeletal muscle (SKM) in human and mouse. *P < 0.05. **Two normal SKM sampleshad undetectable levels of EphrinB2. (B) Representative images of human aRMS from tumor microarray stained for EphB4. 0 represents <10% cells positive.(Scale bar, 50 μm.)

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Fig. S3. Immunohistochemistry for PDGFRβ on a human aRMS tissue microarray in (A) and for EphB4 and PDGFRβ on normal human muscle in (B). (A)Representative images of human aRMS from tumor microarray stained for PDGFRβ. 0 represents <10% cells positive. (Scale bar, 50 μm.) (B) Representativeimages of human SKM stained for EphB4, (C) and for PDGFRβ. (Scale bars, 25 μm.)

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Fig. S4. Immunohistochemistry of an aRMS tumor for receptor-ligand combinations. Representative images of human aRMS section express EphB4 and itscognate ligand, EphrinB2, PDGFRβ, and ligand PDGF-BB. Sections were simultaneously incubated with primary antibody preincubated with respective blockingpeptide to confirm specificity of staining. Images were taken at 40× magnification. (Scale bars, 50 μm.)

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Fig. S5. Immunohistochemistry of a second, independent aRMS tumor for receptor-ligand combinations. Representative images of human aRMS sectionsexpress EphB4 and its cognate ligand, EphrinB2 as well as PDGFRβ, and ligand PDGF-BB. Sections were simultaneously incubated with primary antibodypreincubated with respective blocking peptide to confirm specificity of staining. Images were taken at 40× magnification. (Scale bars, 50 μm.)

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Fig. S6. Effects of knockdown on Pax3:Foxo1, EphB4 and Pdgfrß on transcription of themselves and reciprocal genes in mouse and human aRMS. (A) Pax3:Foxo1 silencing by eYFP targeted siRNA in U23674 and U48484 murine aRMS results in a decrease and increase in EphB4 and Pdgfrβ mRNA levels, respectively.(B) Silencing PAX3:FOXO1 in Rh5 and Rh30 results in a decrease and increase in EPHB4 and PDGFRβ mRNA levels. (C) siRNA targeting EphB4 does not result ina significant increase in Pdgfrβ mRNA levels; however, silencing Pdgfrβ results in a decrease in EphB4 mRNA levels in U23674 and U48484 murine aRMS cells.Identical results are observed in (D) the human aRMS cell line Rh5. *P < 0.05 compared with nonspecific siRNA by Student t test. (E) Silencing Pdgfrα, c-Met,EphB4, and Pdgfrβ in U23674 after Pax3:Foxo1 knockdown. Nonspecific siRNA and siRNA targeting plk1 included as controls. *P < 0.05 by Student t test.Because EphB4 and Pdgfrβ expression were both modulated after silencing of Pax3:Foxo1, we surmised that these may represent downstream effectors ofPAX3:FOXO1 that are especially vital to aRMS cell survival even if PAX3:FOXO1 could be inhibited. To test this hypothesis, we performed combination siRNAexperiments where we first transfected U23674 cells with nonspecific or eYFP siRNA. Two days later, we transfected cells with siRNA targeting Pdgfrβ, EphB4,Pdgfrα (previously shown to be a direct transcriptional target of PAX3:FOXO1), c-Met (previously shown to be a direct transcriptional target of PAX3:FOXO1),Plk1 (target in an unrelated pathway), or nonspecific siRNA. Four days after this second siRNA transfection, cell viability was assessed. Relative viability of cellssubjected to siRNA targeting each gene in combination with eYFP was normalized to cells treated with eYFP plus nonspecific siRNA. Relative viability of cellssubjected to siRNA targeting each gene in combination with nonspecific siRNA was normalized to cells treated with successive exposure to nonspecific siRNA.As expected, silencing of Pax3:Foxo1 alone also resulted in a decrease in tumor cell viability.

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Fig. S7. Pax3:Foxo1 drives EphB4 expression at both the mRNA and protein level. (A) C2C12 cells (murine myoblasts) were transiently transfected with anempty vector (pcDNA3) or a vector expressing Pax3:Foxo1 (pcDNA3-Pax3:Foxo1). Subsequently EphB4mRNA levels were analyzed by RT-qPCR showing that thefusion transcript does drive EphB4 transcript and (B) protein expression and the transfected C2C12 cells were confirmed to express Pax3:Foxo1 at the proteinlevel compared with cells transfected with empty vector as described above. *P < 0.01. (C) Small-molecule inhibitors are able to abrogate PDGF-BB–mediatedgrowth of U23674 cells: PDGF-BB acts as a mitogenic factor for U23674 cells when comparing untreated and vehicle (DMSO) treated cells to PDGF-BB–treatedcells. Both imatinib and dasatinib are able to inhibit this PDGF-BB–mediated growth.

Fig. S8. Relative cell growth of dasatinib-treated aRMS and cell cycle analysis of transfected murine aRMS. (A) Human aRMS Rh5 and Rh30 treated withincreasing concentrations of dasatinib for 72 h and cell viability assessed by MTS assay demonstrate absolute IC50 values at ∼35 nM and∼200 nM, respectively.(B) FACS analysis of cell cycle after cells transiently transfected with siRNA targeting EphB4 or Pdgfrβ. Two days posttransfection, cells were trypsinized, fixed in70% ethanol for 10 min, then stained with PI/RNase for 5 min before FACS analysis with BD FACS Aria III. Data shown is representative from two individuallyperformed experiments. Data analyzed by FlowJo 7.6.5 (Tree Star, PC v7.6). (C) Anchorage-independent cell growth of mouse aRMS after Pdgfrβ or EphB4silencing. 10× objective lens images taken show anchorage independent colony formation in soft agar by U23674 and U48484 after silencing Pdgfrβ or EphB4compared with nonspecific siRNA are significantly decreased in number. See quantification in Fig. 4D.

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Fig. S9. Proposed model of EphB4 signaling in aRMS. The mechanism of EphB4 activation determines the resulting cellular effect. EphB4 phosphorylationmediated by the receptor tyrosine kinase PDGFRβ, when activated by PDGF-BB, activates EphB4 to signal through pathways promoting cell proliferation andsurvival. In the context of EphB4 “forward” signaling through its cognate ligand, EphrinB2, EphB4 activation results in downstream Crkl activation, whichpotentially mediates tumor cell apoptosis. It should be noted that although we observed exogenous EphrinB2 treatment resulted in induction of apoptosis intumor cells, as has been observed before (1), our studies have not yet taken into account the manner by which EphrinB2 is expressed by cells of the tumormicroenvironment, particularly of the vasculature (2), and in this capacity may interact with tumor cells expressing EphB4. Cell–cell interaction and relatedforward or reverse signaling of the EphB4-EphrinB2 axis in a complete tumor microenvironment warrants further study.

1. Noren NK, Foos G, Hauser CA, Pasquale EB (2006) The EphB4 receptor suppresses breast cancer cell tumorigenicity through an Abl-Crk pathway. Nat Cell Biol 8(8):815–825.2. Noren NK, Lu M, Freeman AL, Koolpe M, Pasquale EB (2004) Interplay between EphB4 on tumor cells and vascular ephrin-B2 regulates tumor growth. Proc Natl Acad Sci USA 101(15):

5583–5588.

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