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.

S8Cell145,926–940,June10,2011ª2011ElsevierInc.

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.

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