(D)RT-PCR:mRNAlevelsofZEB2relativetountreatedcontrol:concomitantknockdownofSFRP1(shSFRP1)andadditionofofanti-DKK1(10mg/ml)antibodyallowBMP-antagonistGremlin(2.5ug/ml)orWnt5a(250ng/ml)topromoteTGF-b-inducedexpressionofEMT-TFZEB2.(E)Brightphasemicroscopy:imagesofuntreatedcontrolcells(HMLE24+-shGFPand-shSFRP1),HMLE24+-shGFPcellstreatedwithTGF-b1(2.5ng/ml),HMLE24+-shSFRP1cellstreatedwiththeEMTinductioncocktail(IC):TGF-b1(2.5ng/ml),Wnt5a(250ng/ml),anti-DKK1-(10mg/ml)andanti-E-cadherin-antibody(1mg/ml).Factorswereaddedevery48h.
(F)Growthcurves:proliferationofphenotypicallystable,indicatedcelllineswasmonitoredbytheMTSassayfor3d,8passagesaftercessationofa14dtreatmentasdescribedin(E),n=12.
(G)RT-PCR:mRNAexpressionofZEB1,ZEB2,E-cadherinandN-cadherin,allrelativetoHMLE24+-shGFPcontrolcells.ShownarePBS-treatedcontrolHMLE24+-shGFPandHMLE24+-shSFRP1cells,2replicatesofHMLE24+-shGFPculturestreatedwithTGF-b1(aandb),and2replicatesofHMLE24+-shSFRP1culturestreatedwiththeIC(aandb).mRNAwasisolated8passagesaftercessationofa14dtreatmentasdescribedin(E).(H)Luciferasereporterassay:indicatedcellsweretransfectedwithsmad(SBE4)andb-catenin/TCF-LEF(TOPFLASH)reporterplasmids,reporterassayswereconducted8passagesaftercessationof14dtreatmentasdescribedin(E),n=3.
(I)Immunoblot:EMTmarkersandassociatedautocrinepathways,proteinwasextracted8passagesaftercessationof14dtreatmentasdescribedin(E).Dataarepresentedasmean±SEM.
S12Cell145,926–940,June10,2011ª2011ElsevierInc.
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FigureS6.CharacterizationofPrimaryMammaryEpithelialCellPopulationsthatAreEnrichedinSelf-RenewalandMotility,RelatedtoFigure6
(A)FACS:single-cellpreparationswereobtainedfromhumanreductionmammoplasties;(1.)deadcellswereexcludedby7AADstaining,followedby(2.)exclusionofcellspositiveforCD45(whitebloodcells)andCD31(endothelialcells).(3.)UsingtheresultantLin-cells,MECEPC-(CD49f+/EpCAM-)andMECEPC+(CD49f+/EpCAM+)populationswerecollected.
(B)Brightphasemicroscopy:FACS-puri edMECEPC-,MECEPC+andunsortedMECwereculturedfor5d.
(C)RT-PCR:mRNAexpressionofClaudin-2(CLDN2),ZO-1andE-cadherin(E-cad)inculturedMECEPC-andMECEPC+5dafterFACS.
(D)RT-PCR:mRNAexpressionofp63andEMTTFsinculturedMECEPC-andMECEPC+5dafterFACS.(E)Mammosphereassay:MECEPC-migratedcellswerecollectedfromthebottomofBoydenChambersbytrypsinizationandtheirmammosphereformingabilitycomparedtoparentalMECEPC-andMECEPC+,n=24wells.(F)Immuno uorescence:puri edMECEPC-,MECEPC+culturedfor5dafterFACS:differentialexpressionofmyoepitheliallineagemarkeralpha-smoothmuscleactin(alpha-SMA,expressedin<1/100cells/well),basallineagemarkerscytokeratin14(CK14)andp63.Differentialexpressionofluminallineagemarkersmucin1(MUC1),cytokeratin8(CK8),CK18.
(G)Immuno uorescence:membranousandcytoplasmic/nuclearlocalizationofb-catenininculturedMECEPC-andMECEPC+,5dafterFACS.(H)RT-PCR:Axin2mRNAexpressioninculturedMECEPC-andMECEPC+,5dafterFACS.(I)Growthcurve:MECEPC-treatedevery24hwithindicatedfactors:TGF-breceptorI(TGFBRI)-kinaseinhibitorsA83-01andSB43(1542)(10mM),SFRP1(1mg/ml);cellswerecountedatindicatedtimepoints,n=2.Dataarepresentedasmean±SEM.
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FigureS7.WntandTGF-bSignalsGovernInterconversionsbetweenSC-Enriched,Basal,andLineage-Rrestricted,LuminalMEC,RelatedtoFigure7
(A)Mammosphereassay:MECEPC-wereseededintothemammosphereassayaftera5dpre-treatmentwithTGF-breceptorI(TGFBRI)-kinaseinhibitorsA83-01(3mM)andrecombinantSFRP1(300ng/ml)oracombinationofboth(A+S)inmonolayerculture,inhibitorswereaddedevery24h,n=12wells.
(B)RT-PCR:mRNAexpressionofSlug,Claudin-2(CLDN2)andp63inMECEPC-followingtreatmentasdescribedin(A),andMECEPC+,allrelativetountreatedcontrolMECEPC-.
(C)Immuno uorescence:expressionofE-cadherin,ZO-1,Slugandp63inMECEPC-2daftera5dtreatmentasdescribedin(A).
(D)Mammosphereassay:MECEPC+wereseededintothemammosphereassayaftera5dpretreatmentinmonolayerculturewiththefollowingfactors,addedsinglyorinindicatedcombination:recombinantTGF-b1(TGF-b,0.1ng/ml),anti-E-cadherinantibody(aEC,2mg/ml),amixofWnt-pathwaystimulatingfactorsreferredtoasWNT(anti-DKK1antibody(10mg/ml),anti-SFRP1antibody(1mg/ml),recombinantWnt5a(250ng/ml),n=10wells.(E)MigrationAssay:FACSpuri edMECEpCAM+werepretreatedfor5dasdescribedin(D),shownaremigratedcells/ eld,5 eldswerecounted,n=3wells.(F)GrowthcurveofMECEPC+duringtreatmentwithfactorsdescribedin(D),n=2.(G)GrowthcurveofMECEPC+duringtreatmentwithfactorsasdescribedinFigure7A,n=2.Dataarepresentedasmean±SEM.
S16Cell145,926–940,June10,2011ª2011ElsevierInc.
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