上皮-间质转化信号通路研究获进展 (Cell, 2011)-part2

FigureS2.EMT-AssociatedAutocrineSignaling,RelatedtoFigure2

(A)RT-PCR:mRNAexpressionofBMPligandsinHMLE24+,HTwistandMSPcells,n=3.

(B)Luciferasereporterassay:Smadtranscriptionalactivity:cellsweretransfectedwithSBE4-lucreporterplasmid, re yluciferaselevelswerenormalizedtopGL-SV40renillatransfectioncontrol;cellsweretreatedwithrecombinantTGF-b1(5ng/ml)for30min,n=3.

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(C)RT-PCR:mRNAexpressionofSFRPisoformsinHMLE24+andMSPcelllines,n=3.

(D)RT-PCR:mRNAexpressionofWntligandsinHMLE24+,HTwistandMSPcelllines,n=3.(E)SummaryofdifferentialexpressionofsecretedproteinsregulatingWnt,TGF-bandBMPpathwaysinHMLE24+,HTwistandMSPcelllines.Dataarepresentedasmean±SEM.

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FigureS3.AutocrineSignalingControlsMigrationandMammosphereFormation,RelatedtoFigure3

(A)Growthcurves:proliferationassay(MTS)ofMSPandHTwistcelllinestreatedevery24hforadurationof4dwithrecombinantDKK1(1mg/ml),SFRP1(1mg/ml)orBMP4protein(0.5mg/ml),n=4.

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(B)Immunoblot:inhibitionofconstitutiveSmad2phosphorylationinMSPcellsbyTGFBRI-kinaseinhibitorsA83-01andSB43(1542),addedataconcentrationof10mMfor30min.Loadingcontrol:totalSmad2/3.

(C)Immuno uorescence:inhibitionofnucleartranslocationofSmad2/3byA83-01orSB43(10mM)inHMLE24+cellstreatedconcomitantlywith2.5ng/mlTGF-b1,cellswere xed30minafteradditionoffactors.

(D)MammosphereAssay:HTwistandMSPweretreateddailyduringmammosphereformation(5d)withBMP4(0.5mg/ml),SB43(10mM)oracombinationofboth(B+S),n=6wells.

(E)MigrationAssay:HTwistandMSPcellswereseededintoBoydenChambersinthepresenceofA83-01andSB43(10mM),n=3wells.(F)Bright eldmicroscopyimagesofHMLE-Twist-ERuntreatedcontrolcells(-HTX),andtreatedwith20nMhydroxytamoxifendailyfor12d(+HTX).(G)Mammosphereassay:HMLE-Twist-ERcellstreatedwithHTXfor12danduntreatedcontrolcellswereplatedinmammosphereassaysintheabsenceoffurtherHTXtreatment.SFRP1(1mg/ml)orBMP4protein(0.5mg/ml)wereaddedevery24hsinglyorincombination(S+B)duringmammosphereformation(5d),n=8wells.

(H)Migrationassay:HTX-treatedandcontrolHMLE-Twist-ERcellswereplatedinBoydenChambersandtreatedanalogousto(G),n=3wells.Dataarepresentedasmean±SEM.

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ByaceoiFigureS4.AutocrineSignalingControlsTumorigenicityandMetastasis,RelatedtoFigure4

(A)Flowcytometry:CD44-APCandCD24-PEcellsurfacemarkerexpressioninRAS-transformedHMLE24+,HTwistandMSPcells.Numbersindicate%oftheCD44hi/CD24-population.

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(B)Tumorigenicityassay:HMLE24+-RAS,HTwist-RAS,MSP-RAS,orHMLE24+andMSPcellswereimplantedsubcutaneouslyatindicatednumbersinmice.Micewereeuthanizedandnecropsiedafter10weeks.

(C)Lungmetastasis: uorescentimagesofGFP-positivemetastaticfoci:HMLE24+-RAS,MSP-RASandindependentlyisolatedandtransformedMSP-II-RAScellswereimplantedinthefatpadsofmice.

(D)Quanti cationoflungsurfacemetastaticfoci,shownaremetastases/lunglobe,n=5mice/group.(E)Lungcolonizationassay: uorescentimagesofGFP-positivemetastaticfoci:HMLE24+-RASandMSP-RAScellswereinjectedviatailvein.(F)Histologyofmacroscopiclungmetastases,hematoxylinandeosinstaining.Tumorcellshavemuchlargernucleicomparedtomurinelungepithelium:HMLE24+-RAScellsformductalstructures,MSP-RAScellsgrowdiffusivelywithnodistinctbordertolungepithelium.(G)Quanti cationoflungsurfacemetastaticfoci,shownaremetastases/lunglobe,n=10mice/group.

(H)ProliferationAssay.MSP-RAScellsweretreateddailyfor5dwithSFRP1(1mg/ml),BMP4(0.5mg/ml),proteinoracombinationofboth,cumulativeproliferationover5dwasmeasuredbytheMTSassay,n=6.

(I)Flowcytometry:CD44-APCandCD24-PEcell-surfacemarkersofdissociatedprimarytumorspheres(aftertreatmentasdescribedinFigure4B).Numbersindicate%ofCD44hi/CD24-(upperrightquadrant)andCD44-med/CD24+(lowerrightquadrant).

(J)Histologyofprimarytumorsfromdissociatedprimarytumorspheres(aftertreatmentasdescribedinFigure4B):nomajordifferencesintumorhistologywereobservedbetweendifferentgroups.

Dataarepresentedasmean±SEM.

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RByahcetoiFigureS5.CreationofaPermissiveExtracellularEnvironmentforEMT-Induction,RelatedtoFigure5

(A)Immunoblot:Smad2phosphorylationinHMLE24+cells.Proteinwasextraced30minaftertreatmentwithindicatedconcentrationsofrecombinantTGF-b1;loadingcontrol:bÀactin.

(B)RT-PCR:mRNAexpressionofSFRP1inHMLE24+cellsstablyexpressingtwohairpin-encodingvectors(shSFRP1aandshSFRP1b)comparedtocellsex-pressingacontrolhairpintargetingGFP(shGFP),HMLE24+shSFRP1bcellswereusedforEMT-inductionexperiments.

(C)RT-PCR:mRNAlevelsofZEB2relativetountreatedcontrol:concomitantknockdownofSFRP1(shSFRP1)andadditionofanti-DKK1(10mg/ml)antibodyallowTGF-b1(2.5ng/ml)toinduceexpressionofEMT-TFZEB2inHMLE24+cells.

(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.

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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.

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