MQP‐BIO‐JBD‐0011
CharacterizationofSenescenceRegulationbySmurf2andNotch3invitroandinvivo
AMajorQualifyingProjectReport
SubmittedtotheFacultyofthe
WORCESTERPOLYTECHNICINSTITUTE
inpartialfulfillmentoftherequirementsforthe
DegreeofBachelorofScience
in
BiologyandBiotechnology
by
___________________________________LauraFineman
April26,2012
APPROVED:___________________________________ ___________________________________HongZhang,Ph.D. JosephB.Duffy,Ph.D.CellBiology BiologyandBiotechnologyUMassMedicalSchool WPIProjectAdvisorMajorAdvisor
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ABSTRACT
Oneofthemainfunctionsofatumorsuppressoristorepresscelldivision,
sometimesbyinducingcellularsenescence.Smurf2andNotch3havepreviously
beenidentifiedinourlaboratoryastumorsuppressorsinvitro.Itisnotwell
understoodhowthesegenesregulatesenescence.Thepurposeofthisprojectisto
betterunderstandthemechanismsunderlyingthefunctionsofthesegenesin
senescence.AgeneticscreenusingshorthairpinRNAs(shRNAs)wascarriedoutto
identifygenesdownstreamofSmurf2orNotch3inthesenescencepathways.Five
candidategeneswerefurtheranalyzedinfibroblasts.Tocharacterizethefunctionof
Smurf2insenescenceinvivo,aCre‐LoxPsystemwasusedtostudytheconsequence
ofSmurf2overexpressioninmice.IfoundthatSmurf2impactsgrowthand
regulatesp21inyoungmice.
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ACKNOWLEDGEMENTS
IwouldliketothankProfessorHongZhangforgivingmetheopportunityto
joinhislab,andforhissupportandguidancethroughouttheexperimentaland
writingprocess.IwouldliketothankHangCuifordonatinghertimeinhelpingme
becomeproficientatthetechniquesandassaysnecessaryforthecompletionofthis
project.IwouldalsoliketothankYahuiKong,CharusheilaRamkumar,andIvan
Lebedevforteachingmeadditionalskillsthatwereusefulformyproject.Finally,I
wouldliketothankProfessorJosephDuffyforhisguidanceinchoosingagoodlabto
workin,hisassistancewithMQPediting,andhissupportasmyacademicadvisor
forthepastfouryears.
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TABLEOFCONTENTS
SignaturePage…………………………………..……………………………………………………………….1
Abstract……………………………………………………………………………………………………………..2
Acknowledgements…………………………………………………………………………………………….3
TableOfContents……………………………………………………………………………………………….4
Background………………………………………………………………………………………………………..5
MaterialsAndMethods……………………………………………………………………………………..14
Results…………………………………………………………………………………………………….……….22
Discussion………………………………………………………………………………………………………..34
References……………………………………………………………………………………………….………38
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BACKGROUND
ReplicativeSenescence
Normalcells,includingfibroblastcells,inculturereplicateafinitenumberof
timesbeforeenteringsenescence(HayflickandMoorhead,1961).Whenentering
senescence,cellsarepermanentlyarrestedintheG1phaseofthecellcycle,meaning
theydonotenterthereplicativeSphase(Sherwoodetal.,1988).Thisisnottobe
confusedwithquiescentcells,whicharetemporarilyarrestedintheG0phasedueto
ahighcelldensity(Zhang,2007).Senescentcellshaveadistinctmorphology;they
usuallyarelargerthanyoungcells(Bayreutheretal.,1988).Senescentcellsalso
expressanacidicbeta‐galatosidase.WhenstainedatapHof6,thisenzyme
producesabluecoloringthatbecomesdetectableafterabout16hours(Dimrietal.,
1995)
TelomereShortening
Telomeresarestructuresattheendsofchromosomesthatfunctionin
chromosomalprotection(Kimetal.,1994).Theenzymetelomeraseisawayof
maintenanceofthesechromosomalends;however,itisonlyfoundinimmortal
cancercells(Kimetal.,1994).Themajorityofsomaticcellsdonotexpressthis
enzyme(Kimetal.,1994).Asthesesomaticcellsdivide,theamountoftelomeric
DNAdecreases;thislossandeventualdeletionofnecessarysequencesinthe
genomecontributestothefinitereplicationobserved(Harleyetal.,1990).
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SenescencePathways
Telomereshortening,orDNAdamageandcellstressarethetwobigreasons
whycellswillsenesce.Eachsignalinducesaparallelpathway:itisfoundthatDNA
damageinducesthep53‐p21pathway(Herbigetal.,2004)andcellstressinduces
thep16‐RBpathway(BenantiandGalloway,2004).
p53‐p21Pathway
ATM(ataxia‐telangiectasiamutated)andATR(Rad3‐related)areprimary
PI3K‐likeproteinkinasesintheDNAdamagepathway(Herbigetal.,2004).ATM
respondstodouble‐strandbreaksandATRisaprimarymediatorinultravioletlight
damageandstalledreplicationforks(Herbigetal.,2004).Thesekinaseshavebeen
showntophosphorylateandactivatep53,whichinducesreplicativesenescence
(Herbigetal.,2004).
p53affectscellularreplicationbyit’sabilitytobindtoDNA;increased
bindingabilityleadstoreplicativesenescence(Vazirietal.,1997).Disruptionofthe
p53proteinhasbeenshowntoextendproliferationofhumanfibroblastcellsand
p53levelsareshownincreaseinagingfibroblastcells(Vazirietal.,1997).The
increaseofp53isduetoitsphosphorylationbyATMandATRfollowingDNA
damagefromshrinkingtelomeres(Vazirietal.,1997).Increasedp53binding
increasesthetranscriptionofthep21gene,acellcycleregulatorwhoseexpression
hasbeenseentoincreaseinsenescingcells(Herbigetal.,2003).
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p16‐RbPathway
Althoughtelomereerosionisamainpartofthereplicativesenescence
model,otherfactorsalsocontributetothesenescentphenotype(Hütteretal.,
2002).Oxidativedamageisamajorcauseofagingaswell;theoncogeneRASis
activatedafteroxidativedamageoccursandcaninduceprematuresenescence
(Hütteretal.,2002).
ActivatedRASup‐regulatesthecyclin‐dependentkinaseinhibitorp16
(BenantiandGalloway,2004),whoseexpressionisindependentoftelomere
degradationandp53expression(Herbigetal.,2004).p53isabletoinhibitcyclin‐
dependentkinase(CDK)4andCDK6,whoinactivateRb(retinoblastoma)(Ruasand
Peters,1998).Soincreasedlevelsofp16leadtoincreasedlevelsofRb(Ruas,1998).
However,Rb‐inducedsenescenceisnotcompletelyp16dependent;p21hasbeen
showntoup‐regulateRbinthesamewayasp16(Beauséjouretal.,2003).Rb
activationinducessenescencebyrepressionofE2F‐dependentpromoters,leading
toG1cellcyclearrest(Benantietal.,2004).
NotchSignaling
TheNotchsignalingpathwayisnotfullyunderstood,butitconsistsofthe
Notchreceptors,ligands,modifiers,andtranscriptionfactors(Allenspachetal.,
2002).Notch3isoneoffourmammalianNotchreceptors;itdiffersfromNotch4in
thatitcontainscytokineresponse(NCR)regionsanddiffersfromNotch1and
Notch2inthatitlacksC‐terminaltranscriptionalactivationdomains(TAD)
(Allenspachetal.,2002).
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NotchSignalingPathway
Notchsignalingstartswithbindingtotheligand;thisreleasestheactive
intracellulardomainofNotch(ICN)intothecytoplasm(Allenspachetal.,2002).
Fromthecytoplasm,itrelocatestothenucleus,whereitinducestranscriptionof
severalbasic‐helix‐loop‐helix‐orangeproteinsthatrepressgeneexpression
(Allenspachetal.,2002).Oneofthemajorplayersintranscriptionalactivationisthe
Mastermind‐like(MAML)protein;itaidsinthestabilizationoftheDNA‐binding
complexthatincludesICN(Oyamaetal.,2007).
Notch3andPathways
IthasbeenshownthatNotch3depletionofthecelldecreasedthelevelsof
p21;thiswasconsistentwiththedecreasedlevelsoftheICNtarget,HES1
(Giovanninietal.,2009).HES1increasehasbeenpreviouslylinkedwithup‐
regulationofp21(Giovanninietal.,2009).
Smurf2RegulatesSenescence
Smurf2,orSMADspecificE3ubiquitinproteinligase,encodesa748amino
acidhumanproteinthatisresponsibleforubiquitinatingSmad2,whichleadstoits
degradation;SmadsareimportantregulatorsforTGF‐β(transforminggrowth
factor‐beta)(Linetal.,2000).
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Smurf2andSenescence
Microarrayanalysisfromourlabhasshownthattelomereshortening
increasesSmurf2levels;furthermore,overexpressionofthegeneisalsosufficientto
inducereplicativesenescence(ZhangandCohen,2004).Twoformsofthisprotein
areusedinthisproject:theabove‐mentionedwildtypeformandacystineto
alaninemutationatposition716intheHECTdomainoftheprotein(Linetal.,
2000).AlthoughtheC716AmutantisunabletoubiquitinateSmad2,itstilloperates
underasimilarmechanismtothewildtype(Zhangetal.,2008).
Smurf2inthePathway
Smurf2hasbeenshowntoaffectboththep53/p21andthep16/Rb
pathways;however,itrequiresatleastoneofthepathwaystobefunctionalinorder
toinducesenescence(ZhangandCohen,2004).Smurf2entersthepathwayat
eitherp21orp16(Figure1).IncreaseinSmurf2hasbeenshowntocorrelatewith
increasedexpressionofp21(Zhangetal.,2008).Smurf2doesnotdirectlyinteract
withp16,Id1(inhibitorofdifferentiationofDNAbinding1)actsasamediator
(Kongetal.,2011).Id1repressesp16expression,sotheubiquitinationand
degradationofId1bySmurf2allowsp16tobeexpressedandinducesenescence
(Zhengetal.,2004).
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Figure1:Notch3’sandSmurf2’sInteractionwiththep53‐p21andthep16‐RBSenescencePathways.Notch3isshowntoup‐regulatep21levels.Smurf2hasbeenshowntobothup‐regulatep21levelsandup‐regulatep16levelsthroughrepressionofId.
IdentificationofGenesDownstreamofNotch3UsingshRNAScreening
TheinvitropartofthisMQPconsistedofdeterminingwhatgeneswere
downstreamofSmurf2andNotch3.Smurf2screeningpreviouslydonebyourlab
hadalreadygeneratedagroupofclonesthatneededtobevalidated,soI
concentratedonNotch3screening.Theideawasifagenethatwasinfact
downstreamofeitherSmurf2orNotch3wasknockeddown,thesenescence
Id
SenescenceSignals
ATM/ATR
p53
p16
p21
RB
E2F
Notch3Smurf2
SenescencePhenotype
RAS
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pathwaywouldbeinterruptedandfibroblastcellswouldbeabletoescapeSmurf2
orNotch3‐inducedsenescence.
TheshRNAlibrariesusedforthescreeningweretheExpressionArrestpGIPZ
Lenti‐viralshRNAmirLibraryfromOpenBiosystems,asshowninFigure2.The
librarywasdividedintopools,eachpoolcontaining5,000shRNAs.Thelenti‐viral
vectoriscompromisedoftwocomplementary22bpsequencesthatwouldpairinto
ahairpinsequencewhentranslatedintomRNA.
Figure2:DesignoftheshRNAmirvector.TheshRNAmirsystemincorporatestwo22bpsequencesthatarecapableofbindingandformingahairpininalenti‐viralconstruct.(AdaptedfromOpenBiosystems)
Thehairpinthatformsactivatesthecellulardrosha/dicerresponse,resulting
inexpressionarrestforthecorrespondingtranscript(Figure3).Droshaanddicer
processthehairpinshRNAmirtoasinglestrand.TheRISCcomplexbindstothe
singlestrandofshRNAmirandthetranscript,allowingdegradationandsilencingof
thegene.
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Figure3:shRNAmirExpressionArrestMechanism.CellsinfectedwithasingleviruscontainingashRNAmirconstructleadstotheexpressionarrestofatranscript.(AdaptedfromOpenBiosystems)
FunctionofSmurf2InVivo
Smurf2ubiquitousexpressionhasnotbeenstudiedmuch;previousinvivo
studiesstudiedtissuespecificexpressionforosteoblastfunction.Inthisproject,we
useamousemodeltostudytheconsequenceofSmurf2overexpression.
Knockout
Previously,theSmurf2knockoutmousemodelwasfirstcreatedbytheZhang
groupattheNationalCancerInstitute.Thismouseshowsgenomeinstabilitythat
leadstoincreasedsusceptibilitytovariouscancers(Blanketal.,2012).Ourlabalso
generatedknockoutmicewithadifferentstrategy,whichhaveasimilarphenotype
(inpress).
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Overexpress
OurlabhascreatedaSmurf2knock‐inmouseusingaCre‐LoxPsystem.A
Rosa26‐LoxP‐STOP‐LoxP‐Smurf2mousewasbackcrossedtoaC57BL/6
background.TheRosa26isastrongpromoterthatiscapableofubiquitous
expressionattheearlyembryonicstage(Soriano,1999).TheLoxPflankingsystem
allowsexpressionofthegeneofinterestuntilrepressedwithaCre‐LoxPsystem
(Rajewskyetal.,1996).Inthiscase,theSTOPisflankedbytheLoxP,andrepresses
theexpressionofSmurf2.ToactivatetheSmurf2gene,themicewerecrossedwith
Sox2‐CremicethatwereabletodisrupttheSTOPsiteandallowexpressionof
Smurf2startingatanearlyembryonicstage.Figure4showsthemechanismthat
occursinSmurf2knock‐inmicewhencrossedwiththeSox2‐Cremice.
Figure4:MechanismofSox2‐Cre‐LoxPsysteminSmurf2knock‐inmice.Smurf2geneandLoxP‐STOP‐LoxPinsertionintoRosa26locusinhibitsexpressionofSmurf2.AdditionofSox2‐CreexcisesSTOPandallowsexpressionofSmurf2.
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MATERIALSANDMETHODS
DNACloning
Cloningwasusedinthisprojectobtainandamplifyplasmidconstructs
neededforDNAtransfection.TheseplasmidsincludedpMD2‐VSVG,pCMVdR8.74,
dsRED,fourdifferentconstructsofCDC42BPA,andfourdifferentconstructsof
AHCYL1.
BacterialCulture
AllplasmidswereobtainedbyXL‐2E.colistockcontainingtheconstruct.The
plasmiddsREDwasculturedunderkanamycinselection;asmallamountofbacteria
wasculturedin6mLofLBmediumcontainingkanamycinforamplificationand
collectedviaminiprep.Allotherconstructswereculturedunderampicillin
selection;asmallamountofbacteriawasstreakedoutontoampicillinplates.Single
colonieswerepickedandculturedin6mLofLBmediumcontainingampicillin.The
CDC42BPAandAHCYL1plasmidswerecollectedviaminiprep.ThepMD2‐VSVGand
pCMVdR8.74constructswereculturedfurtherinpreparationofcollectionvia
midiprep.
PlasmidIsolation
PlasmidDNAsinthisprojectwereisolatedusingQuiagen’sQIAprep®Spin
MiniprepKitandQuiagen’sHiSpeed®PlasmidPurificationKit.
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DNATransfectionin293T
Thisprojectrequiredexpressionofplasmidconstructsincelllines.TheDNA
wasdeliveredinalenti‐viralconstructtointegratetheircontentsintotheinfected
hostcell.Virusespackagedwerelenticontrolvector,Notch3,Smurf2WT,and
Smurf2C716A.Humanembryonickidney293Tcellsfromculturewereplatedata
densityof4.5x106cells/p100tissueculturedishesinDMEMmediumwith10%
fetalbovineserum.Transfectionoccurredthenextdaywith2MCaCl2and2XHBSat
acellconfluenceofabout70%.
TheDNAwasmixedina15mLtube;6.5ugofpackagingvectorpCMVdR8.74,
3.5ugofenvelopevectorpMD2‐VSVG,0.2ugofreportervectordsRED,and10uLof
theDNAofinterest.Thevolumewasraisedto437.5uLwith0.1%TEinH20.Themix
wasvortexedatahighspeed,followedby62.5uLofCaCl2and500uLofHBS,both
addeddropwise.Thefinalsolutionwasaddeddropwiseontothe293Tcells.
Twenty‐fourhourslater,themediumwaschangedto9mLoffreshDMEMwith10%
FBS.Forty‐eighthourspost‐transfection,thecellswereobservedunder
fluorescencemicroscopyforthepresenceofdsRED.Supernatantsofplateswith
morethan70%ofcellsexpressingdsREDwerecollectedandstoredat‐80°Ctokill
anylivingcellscollectedalongwiththevirus.
shRNAScreening
GeneknockdownwithshRNAplasmidsallowedforidentificationofpossible
genesinvolvedinNotch3inducedsenescence.
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CellInfectionwithshRNAVirusandPuromycinSelection
WS1humanfibroblastcellswereplatedat1.0x105cells/p100.Eachp100
platewasinfectedwith5uLofshRNAlibraryinmediumcontaining1ug/uLof
polybrene.Theplateswerechangedwithfreshmediumthefollowingday.The
shRNAplasmidscontainpuromycinresistance;twodayspostinfection,medium
containing1ug/mLofpuromycinwasadded.Ap60platecontainingWS1cellsas
wellwasusedasaselectioncontrol;theplatewasemptyafterabout3daysof
puromycinexposure.Secondroundinfectionoccurredafterselection.
CellInfectionwithNotch3VirusandBSDSelection
WS1cellsthatremainedviableafterpuromycinexposureweretheninfected
withNotch3virusinmediumcontaining1ug/uLofpolybrene.Theplateswere
changedwithfreshmediumthefollowingday.TheNotch3plasmidcontainsBSD
resistance;twodayspostinfection,mediumcontaining1ug/mLofpuromycinand
2ug/mLofBSDwasadded.Ap60containingWS1cellswasagainusedasaselection
control;theplatewasemptyafter4‐5daysofBSDexposure.Thecellswerekept
underpuromycinandBSDselectionfor12days,whencoloniesstartedformingon
theplates.
IsolationofGenomicDNA
GenomicDNAwasthenisolatedfromthecoloniesofthescreenusingspot
trypsinization.Eachcolonywasculturedinanindividualwellona24‐wellplate
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untilalmostreachingconfluence;genomicDNAwasisolatedusingthefollowing
protocol.CellswerewashedwithPBS,andthen200uLofalysisbuffer/5Xprotease
Ksolutionwasadded.Thecellswereincubatedat37°Cfor2‐3hours,andthe
supernatantwascollectedtoa1.5mLEppendorftube.Next,200uLofisopropanol
wasmixedinandthetubewasincubatedfor10‐15minutesatroomtemperature
beforebeingspundownfor15minutesat14,000rpm.Thesupernatantwas
aspiratedandthepelletwaswashedwith70%ethanol;thetubewasspundown
againfor7minutesat14,000rpmandthenairdried.Afterthepelletwasdry,50uL
ofH20wasaddedandthetubewaslefttoincubateovernightat37°C.
PolymeraseChainReaction
TheGenomicDNAextractedfromeachcolonywasamplifiedusing
PolymeraseChainReactionandwasfacilitatedwithTaqDNAPolymerasepurchased
fromNewEnglandBiolabs.TheamplifiedregionincludedtheshRNAmirsequence;
theamplificationproductwassenttobesequenced.
Sequencing
TheDNAPCRproductwaspurifiedandsenttoGenewizInc.forsequencing.
shRNAConformation
ThreeshRNAs(PIAS4,AHCYL1,andCDC42BPA)werevalidatedbytesting
knockdownefficiencyandgrowthcurves.
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RNAIsolation
RNAwasisolatedfromthecellspriortothesecondaryinfectionofNotch3or
Smurf2toidentifythelevelofknockdowneachshRNAinduces.RNAwasisolated
usingaTrizol‐basedprotocol,andthenrunwithgelelectrophoresistodetermine
theintergrityoftheRNA.
RT‐PCR
Invitrogen’sSuperscript®IIReverseTranscriptasewasusedtotranscribe
theextractedRNAintocDNA.
Real‐timeqPCR
ThereversetranscriptaseproductwasrunusingaBio‐RadiQ5machineand
softwarewiththeaidofSYBR®Green.
GrowthCurves
Cellswereplatedin6‐wellplatesataround2x104cells/wellwithtwowells
foreachsampletimepoint.Eachsetofwellswascountedonday1,3,5,and7with
day1beinglessthan20hoursaftercellswereplated.AZ1Coulter®Particle
Counterwasusedtocounteachsamplethreetimes;theaveragewastaken.
Genotyping
Miceweregenotypedandmarkedonthetoeatage7‐10days.
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GenomicDNAExtraction
Approximately2mmoftailwassnippedandplacedinaPCRtubewith75uL
ofNaOHandEDTAlysisbufferandincubatedat95°Cfor30minutesandthen
cooledto4°Cfor15minutes.Then75uLofTris‐HClbufferwasaddedandmixedto
neutralizethesolution.
PolymeraseChainReaction
TheextractedgenomicDNAwasamplifiedusingPolymeraseChainReaction
usingprimersthatsurroundedtheregionofinterest,Smurf2WT,Smurf2C716A,or
Cre.AmplificationwasaidedbyLambdaBiotech’sTaqPolymerase.ThePCRproduct
wasrunona1%agarosegelwithgelelectrophoresistodeterminethegenotypes.
OBSERVATIONOFCONSEQUENCEOFSMURF2INVIVO
ObservationoftheSmurf2overexpressedmicewascarriedoutbyabi‐
weeklyandweeklyrecordofweightaswellasdeterminationofgeneexpressionby
Westernandreal‐timePCR.
GrowthCurve
Growthwasmeasuredfromshortlyafterbirthuntildeath.Weightwas
measuredingramsbyplacingthemouseinabeakerthatwaszeroedonabalance.
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OrganExtraction:RNAandLysateExtraction
Eightorgans;liver,heart,lung,kidney,pancreas,spleen,colon,andskinwere
harvestedandweighed.Eachorganwasfrozeninliquidnitrogenandcrushedinto
powderforsubsequentuseforlysateandRNAextraction.
RNAwasextractedfromthespleenusingtheabovementionedTrizolbased
procedure.TheRNAwasreverse‐transcribedandanalyzedwithreal‐timePCR.
LysatewasextractedfromalltheorgansusingRIPAbufferwith20Xprotease
inhibitor.Concentrationwasmeasuredat562nanometers.Lysateswererunona
WesternblotandprobedforSmurf2,p21,p16,andtubulin.
BoneMarrowExtraction
Theentirebonemarrowextractionprocesswasperformedonice.Micewere
double‐killedusingIsofluraneandcervicaldislocation.Hindlimbswereseveredat
hipjointandforelimbsattheshoulderjoint.Limbswereplacedinap60plate
containingastaining(Biotin‐,Flavin‐,Pheno‐reddeficientRPMI1640)medium.Skin
andmusclewasscrapedawaytothebonewitharazorbladeandthekneeand
elbowjointsweredisarticulatedtoharvestthefemur,tibia,andhumorousbones.
Theendsoftheboneswerecuttoexposethemarrowcavities;boneswere
completelyflushedwithstainingsolutionusinga5mLsyringeanda25Gneedle.An
18Gneedleissubsequentlyusedtobreakthelargechunksofmarrowintosmall
pieces.Cellsarefilteredwitha70umnylonmeshintoa15uLtubeandthefinal
volumeofcellsisbroughtupto10mLwiththestainingsolution.
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A10Xdilutionofcellsiscountedusingahemocytometer.Thebonemarrowwas
extractedforRNAusingthemethodmentionedaboveinprepforrtPCR.
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RESULTS
ProductionofSmurf2andNotch3Viruses
HEK‐293Tcellswereusedtopackagethevirusesusedinthisprojectvia
transfection.AplasmidexpressingaredfluorescentproteindsREDwasusedin
transfectiontodeterminetheefficiencyofthetransfections;Figure5shows
representativeimagesof293Tcellsaftertransfection.StrongerdsREDfluorescence
correlateswithabetterefficiency;efficiencieslowerthat80%werenotused.
Figure5:HEK‐293TTransfectionEfficiency.TransfectionefficiencyisshownbydsREDfluorescence:regularlight(left)andgreenfluorescentlight(right)showstheusualpercentageofHEK‐239Tinfectedbythevirus.
shRNAScreenforGenesInvolvedinNotch3‐InducedSenescence
OurlabhadpreviouslyscreenedseveralcandidatesusingSmurf2,soI
concentratedonscreeningwithNotch3.Sevengeneswereidentifiedfromthe
Notch3screen;Table1showsthegenesidentifiedalongwiththechromosome
locationandthesenseshRNAsequence.CDC42bindingproteinkinasealpha
(CDC41BPA)ishighlightedinthetable;thisgenewasstudiedfurtherinattemptto
confirmthatitoperatesdownstreamofNotch3.BetweenthepreviousSmurf2
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screeningandthecurrentNotch3screen,fivegeneswereanalyzedfurther:SUMO2,
Catalase,PIAS4,AHCYL1,andCDC42BPA.
Pool# GeneName Identity
MatchChromosomeLocation SenseshRNAsequence5’‐>3’
8 Inter‐genicregion 21/22 8 CCGTCTCCAGACATTTAAAGAT
9 Inter‐genicregion 21/22 1 CTGAAGATGTACTGGAACTAAA
9
Glutamatereceptor,ionotropic,kainite3
21/22 1p34‐p33 CCCTTGGTTTCTCCTATGAGAT
10
CDC42bindingprotein
kinasealpha
21/22 1q42.11 CAAGCTGGAAGTTCATACAGAA
11TranscriptionfactorAP‐2
beta21/22 6p12 ACCTCCCTTCCTCACATTGTTA
13 Inter‐genicregion 21/22 14 AGCATGTCACTTGTTCTGTTAA
13 Inter‐genicregion 21/22 20 AGCCTTAAGTTCCACCACACTA
Table1:SevenCandidateGenesThatMayActDownstreamofNotch3.AsummaryofthesequencingdataobtainedfromtheNotch3screening.TheshRNAtargetedgenesareshown,aswellasthenucleotidematchbetweenthegeneandsiRNAandchromosomelocation.CDC42bindingproteinkinasealphawaschosentoresearchfurtherasapossiblecandidate.
TestofKnockdownofCandidateGenes
TotalRNAwasextractedfromcellsinfectedwithlenti‐virusexpressing
shRNAtargetingSUMO2,Catalase,PIAS4,AHCYL1,andCDC42BPA.Afterreverse
transcription,RT‐qPCRwasusedtodetermineiftheshRNAswereabletoinducea
sufficientknockdowninthecells.
SUMO2andCATALASEshRNAwasunabletoproduceanadequate
knockdown(datanotshown),soconfirmationofthesegeneswasdiscontinued.
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BothshRNAstestedinducedmorethan50%knockdownofPIAS4inWS1
fibroblastcells(Figure6)wheninfectedwith200uLofshRNAlenti‐virus.
Figure6:PIAS4_7andPIAS4_9shRNAsProduceaMoreThanFiftyPercentKnockdowninWS1cells.RNAwascollectedfromWS1cellsandreversetranscribedtocDNAforrealtimePCRfromWS1cellstransfectedwith200uLnon‐silencingshRNA(blue),PIAS4_7(red),andPIAS4_9(green).Non‐silencingshRNAwasnormalizedto1.
FourdifferentshRNAsweretestedforAHCYL1,givingvariousknockdowns
fora100uLvirusinfectioninWS1fibroblastcells(datanotshown).ThebestshRNA
wereretestedinLF1cellswith250uLofvirus,givingmorethanan80%knockdown
(Figure7).
‐0.2
0
0.2
0.4
0.6
0.8
1
1.2
KnockdownofPIAS4
NS
PIAS4_7
PIAS4_9
25
Figure7:AHCYL1_4shRNAProducesaMoreThanEightyPercentKnockdowninLF1cells.RNAwascollectedfromLF1cellsandreversetranscribedtocDNAforrealtimePCRfromLF1cellstransfectedwith250uLofnon‐silencingshRNA(blue)andAHCYL1_4(green).Non‐silencingshRNAwasnormalizedto1.
FourdifferentshRNAswerealsotestedforCDC42BPA;thesealsogave
variousknockdownsfora100uLvirusinfectioninWS1fibroblastcells(datanot
shown).ThebesttwoshRNAswereretestedinLF1cellswith250uLofvirus,giving
morethana40%knockdown(Figure8).
0
0.2
0.4
0.6
0.8
1
1.2
KnockdownofAHCYL1
NS
AHCYL1
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Figure8:CDC42BPA(1,7)shRNAProduceaMoreThanFortyPercentKnockdowninLF1cells.RNAwascollectedfromLF1cellsandreversetranscribedtocDNAforrealtimePCRfromWS1cellstransfectedwith250uLofnon‐silencingshRNA(blue),CDC42BPA_1(red),andCDC42BPA_7(green).Non‐silencingshRNAwasnormalizedto1.
ValidationofPIAS4,AHCYL1,andCDC42BPA
Inordertoensurethatthecandidatesthatwerescreenedwerenot
background,growthcurveswereused.WS1orLF1fibroblastcellswithnon‐
silencing,PIAS4,AHCYL1,andCDC42BPAshRNAwereinfectedwithlenti‐virus
controlvectorandeitherSmurf2WTaswellasSmurf2C716AorNotch3challenge
viruses.Thecellswereplatedfourdayspost‐infection(Notch3)orfivedayspost‐
infection(Smurf2WTandC716A).
Figure9belowshowsthegrowthcurvedatafromPIAS4_7andnon‐silencing
knockdowncellswithalenti‐viruscontrolvector,Smurf2WT,orSmurf2C716A
challengeinWS1cells.ThedatashowsthatPIAS4/lenticontrolgrowthrateisabout
thesameasnon‐silencing/lenticontrol.ThedataalsoshowsthatPIAS4knockdown
(dashedblueandred),whenchallengedwithSmurf2WTandSmurf2C716A,does
0
0.2
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0.6
0.8
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Knockd
ownofCDC
42BP
A
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CDC42BPA_1
CDC42BPA_7
27
notleadtoasignificantrescueofsenescencewhencomparedtonon‐silencing
knockdown(solidblueandred).
Figure9:PIAS4_7KnockdownDoesNotLeadtoSignificantRescueofSmurf2WTandSmurf2C716AInducedSenescence.CellgrowthdatawascollectedfromLF1cellstransfectedwithnon‐silencingshRNA/lenti‐viruscontrolvector(solidgreen),non‐silencingshRNA/Smurf2WT(solidblue),non‐silencing/Smurf2C716A(solidred),PIAS4/lenti‐viruscontrolvector(dashedgreen),PIAS4/Smurf2WT(dashedblue),andPIAS4/Smurf2C716A(dashedred).DatashowsthatPIAS4cellschallengedwitheitherSmurf2WTorSmurf2C716Aarenotrescuedfromthecontrolsenescencephenotypeshownbynon‐silencingshRNA/Smurf2WTandnon‐silencingshRNA/Smurf2C716A.
Figure10belowshowsthegrowthcurvedatafromAHCYL1andnon‐
silencingknockdowncellswithalenti‐viruscontrolvector,Smurf2WT,orSmurf2
C716AchallengeinLF11cells.ThedatashowsthatAHCYL1/lenticontrolgrowth
rateisslowerthannon‐silencing/lenticontrol.ThedataalsoshowsthatAHCYL1
knockdown(dashedblueandred),whenchallengedwithSmurf2WTandSmurf2
C716A,doesnotleadtoasignificantrescueofsenescencewhencomparedtonon‐
silencingknockdown(solidblueandred).
0
100000
200000
300000
400000
500000
0 1 2 3 4 5 6 7 8
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ber
DaysinCulture
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NS+S
NS+C
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P+S
P+C
28
Figure10:AHCYL1KnockdownDoesNotLeadtoSignificantRescueofSmurf2WTandSmurf2C716AInducedSenescence.CellgrowthdatawascollectedfromLF1cellstransfectedwithnon‐silencingshRNA/lenti‐viruscontrolvector(solidgreen),non‐silencingshRNA/Smurf2WT(solidblue),non‐silencing/Smurf2C716A(solidred),AHCYL1/lenti‐viruscontrolvector(dashedgreen),AHCYL1/Smurf2WT(dashedblue),andAHCYL1/Smurf2C716A(dashedred).DatashowsthatAHCYL1cellschallengedwitheitherSmurf2WTorSmurf2C716Aarenotrescuedfromthecontrolsenescencephenotypeshownbynon‐silencingshRNA/Smurf2WTandnon‐silencingshRNA/Smurf2C716A.
Figure11belowshowsthegrowthcurvedatafromCDC42BPA_1andnon‐
silencingknockdowncellswithalenti‐viruscontrolvectororNotch3challengein
LF1cells.ThedatashowsthatCDC42BPA/lenticontrolgrowthrateisfasterthan
non‐silencing/lenticontrol.ThedataalsoshowsthatCDC42BPAknockdown
(dashedblue),whenchallengedwithNotch3,doesnotleadtoasignificantrescueof
senescencewhencomparedtonon‐silencingknockdown(solidblue).
0
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40000
60000
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100000
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200000
0 1 2 3 4 5 6 7 8
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ber
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NS+S
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A4+V
A4+S
A4+C
29
Figure11:CDC42BPA_1KnockdownDoesNotLeadtoSignificantRescueofNotch3InducedSenescence.CellgrowthdatawascollectedfromLF1cellstransfectedwithnon‐silencingshRNA/lenti(solidgreen),non‐silencingshRNA/Notch3(solidblue),CDC42BPA/lenti(dashedgreen),andCDC42BPA/Notch3(dashedblue).DatashowsthatCDC42BPAcellschallengedwithNotch3arenotrescuedfromthecontrolsenescencephenotypeshownbynon‐silencingshRNA/Notch3.
Figure12belowshowsthegrowthcurvedatafromCDC42BPA_7andnon‐
silencingknockdowncellswithalenti‐viruscontrolvector,Smurf2WT,orSmurf2
C716AchallengeinLF11cells.ThedatashowsthatCDC42BPA/lenticontrolgrowth
rateisslowerthannon‐silencing/lenticontrol.ThedataalsoshowsthatCDC42BPA
knockdown(dashedblueandred),whenchallengedwithSmurf2WTandSmurf2
C716A,doesleadtoasignificantrescueofsenescencewhencomparedtonon‐
silencingknockdown(solidblueandred).
0
50000
100000
150000
200000
250000
300000
350000
0 1 2 3 4 5 6 7 8
Cell#
Day#
NS+V
NS+N3
C+V
C+N3
30
Figure12:CDC42BPA_7KnockdownDoesLeadtoSignificantRescueofSmurf2WTandSmurf2C716AInducedSenescence.CellgrowthdatawascollectedfromLF1cellstransfectedwithnon‐silencingshRNA/lenti‐viruscontrolvector(solidgreen),non‐silencingshRNA/Smurf2WT(solidblue),non‐silencing/Smurf2C716A(solidred),CDC42BPA/lenti‐viruscontrolvector(dashedgreen),CDC42BPA/Smurf2WT(dashedblue),andCDC42BPA/Smurf2C716A(dashedred).DatashowsthatCDC42BPAcellschallengedwitheitherSmurf2WTorSmurf2C716Aarerescuedfromthecontrolsenescencephenotypeshownbynon‐silencingshRNA/Smurf2WTandnon‐silencingshRNA/Smurf2C716A.
SenescenceRegulationbySmurf2InVivo
ToanalyzewhathappenswhenSmurf2isoverexpressedinmice,we
weighedthemiceeveryonetotwoweeksandcheckedSmurf2,p21,andp16
expressionlevelsinorgansandbonemarrow.
Thereweresevenmiceinthelitter:threewereSox2‐CreandfourwereSox2‐
Cre/Smurf2.Forthefirstmonth,themicewereweighteveryotherweek;theywere
weighedeveryweekforthesecondmonth(Figure13).Thedatashowsthatthe
Sox2‐Cre/Smurf2(blue)miceweighlessthantheSox2‐Cre(red)micefor
approximatelythefirstfortydays.
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ber
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NS+V
NS+S
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C+C
31
Figure13:Smurf2OverexpressingMiceWeightLess.Micewereweighedeverytwoweeksandtheneveryweekfortwomonths.Thelitterwassevenmice:threewereSox2‐Cre(red)andfourwereSox2‐Cre/Smurf2(blue).
Todetermineproteinexpression,lysatewasextractedfromeightorgansand
runonaWesternblot.Theorgansextractedwereliver,kidney,spleen,pancreas,
colon,lung,heart,andskin.Themembranewasprobedwiththreeantibodies:
Smurf2,p21,andp16,butp21andp16wereundetected.AsseenwithFigure14,
Smurf2proteinisoverexpressedinSox2‐Cre/Smurf2micewhencomparedtoSox2‐
Cremice.
liverkidneyspleenpancreascolonlungheartskin‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐5858585858585858
Figure14:Smurf2ProteinisOverexpressedinSox2‐Cre/Smurf2Mice.WesternblotofSox2‐Cremouse#5andSox2‐Cre/Smurf2mouse#8organs.Organsincludeliver,kidney,spleen,pancreas,colon,lung,heart,andskin.MembranewasprobedwithSmurf2.
0
5
10
15
20
25
30
35
0 10 20 30 40 50 60
Weight(gram
s)
DaysafterBirth
Sox2‐Cre/Smurf2
Sox2‐Cre
Smurf2tubulin
32
ThegeneexpressionofSmurf2,p21,andp16wasdeterminedusinganassay
moresensitivethanaWesternblot.RNAwasextractedfromspleenandanalyzed
usingreal‐timePCR.AsseeninFigure15,Smurf2andp21RNAisoverexpressedin
Sox2‐Cre/Smurf2(blue)micewhencomparedtoSox‐Cre(red)mice.Thedataalso
showsthatp16isnotoverexpressedinSox2‐Cre/Smurf2mice.
Figure15:Smurf2andp21RNAisOverexpressedinSox2‐Cre/Smurf2mice.RNAwasextractedfromspleeninSox2‐Cre(blue)andSox2‐Cre/Smurf2(red)mice.ExpressionofSmurf2,p21,andp16wasanalyzed.Sox2‐Cregeneexpressionwasnormalizedto1.
ThegeneexpressionofSmurf2,p21,andp16RNAwasalsodeterminedwith
bonemarrowandanalyzedusingreal‐timePCR.AsseeninFigure16,Smurf2RNAis
overexpressedinSox2‐Cre/Smurf2(blue)micewhencomparedtoSox‐Cre(red)
mice.Thedataalsoshowsthatp21isnotsignificantlyoverexpressedinSox2‐
Cre/Smurf2miceandthatp16expressionisundetectable.
0
1
2
3
4
5
6
7
RelativeGeneExpression
Smurf2p21p16
Sox2‐Cre
Sox2‐Cre/Smurf2
33
Figure16:Smurf2RNAisOverexpressedinSox2‐Cre/Smurf2mice.RNAwasextractedfrombonemarrowinSox2‐Cre(blue)andSox2‐Cre/Smurf2(red)mice.ExpressionofSmurf2,p21,andp16wasanalyzed.Sox2‐Cregeneexpressionwasnormalizedto1.
0
0.5
1
1.5
2
2.5
RelativeGeneExpression
Smurf2p21p16
Sox2‐Cre
Sox2‐Cre/Smurf2
34
DISCUSSION
Discussion
ThisprojectwassuccessfulinusingshRNAmirgeneknockdownscreeningto
identifypotentialgenesdownstreamofNotch3.Sevencandidategeneswere
identifiedfromthisscreeningprocess,oneofwhichwasfurtherinvestigatedwith
fivegenesintheSmurf2senescencepathway.Mydatasuggeststhatknockdownof
CDC42BPAallowspartialescapeofSmurf2inducedsenescence,whileknockdown
ofCDC42BPAdoesnotallowsignificantrescuefromNotch3‐inducedsenescence
andknockdownofPIAS4andAHCYL1doesnotallowsignificantrescuefrom
Smurf2‐inducedsenescence.
Thisprojectwasalsosuccessfulindeterminingpreliminarycharacterization
ofthefunctionofSmurf2insenescenceregulationinvivo.Smurf2overexpressionis
abletoimpactgrowthandregulatep21inyoungmice.
PIAS4,AHCYL1,andCDC42BPA
PIAS4,AHCLY1,andCDC42BPAwereidentifiedascandidategeneswhose
down‐regulationallowstheescapeofSmurf2‐orNotch3‐inducedsenescencein
WS1andLF1cells.
PIAS4
IthasbeensuggestedpreviouslythatPIAS4isinvolvedinsenescence
(Rytinkietal.,2009).PIAS,orproteininhibitorsofactivatedSTATs,isabletoinhibit
STATasitsnamesuggests(Desrivièresetal.,1996).AsSTATactivatestranscription,
35
PIASisabletoinhibittranscription(Desrivièresetal.,1996).PIAS4(PIASyor
gamma)isshowntoactasaSUMO‐E3ligaseforSmad3andp53(Imotoetal.,2003).
MyresultsindicatethatknockdownofPIAS4isnotsufficienttoallow
fibroblaststoescapeSmurf2‐inducedsenescence.Itispossiblethatthelevelof
knockdowninmyexperimentswasnotdepletedenoughtointerferewiththe
functionofPIAS4,orthatotherPIASproteins(PIAS1‐3)compensateforthe
knockdownofPIAS4.Inthecaseofcompensation,afunctioninterferenceofPIAS
activitywouldbemoresuitableinthetestoftheirfunctioninSmurf2‐induced
senescence.ItisalsopossiblethatknockdownofPIAS4onlycompromisesonlyone
ofthesenescencepathways,whichisnotsufficientforafullescapeofSmurf2‐
inducedsenescence.ItwillbeinterestingtoinvestigateifPIAS4collaborateswith
othergenesinregulatingSmurf2‐inducedsenescence.
AHCYL1
ItisknownthatAHCYLcanregulatebothsenescencepathwaysthroughp53
andp16(Lealetal.,2008).AHCYL1,ors‐adenosylhomocysteinehydrolase‐like1,is
involvedinthehydrolysisofS‐adenosyl‐L‐homocysteinetoL‐homocysteineand
adenosine(Lealetal.,2008).AlthoughthereislittleresearchonAHCYL1in
particular,inactivationofAHCYLingeneralinducedcellresistancetop53andp16‐
inducedreplicativearrest(Lealetal.,2008).AHCYLhasalsobeenshowntoinhibit
p53transcriptionalactivityandDNAdamage‐inducedtranscriptionofp21(Lealet
al.,2008).
36
MydatagivesevidencethatknockdownofAHCYL1isnotsufficienttoresuce
fibroblastsfromSmurf2‐inducedsenescence.AsverygoodknockdownofAHCYL1
wasachieved,itislikelythatthelevelofAHCYL1knockdowninterferedwithits
function.ApossibilityisthatAHCYL2wasabletocompensatefortheknockdownof
AHCYL1,soagain,afunctioninterferenceofAHCYLactivitymightbemoresuitable
intestingitsfunction.AnalternativereasonisthatAHCYL1knockdowncausesthe
WS1andLF1fibroblastcellstogrowveryslowly,evenwithoutthesecondary
infection;fastergrowthwasunabletobeachieved.SoalthoughAHCYL1knockdown
withSmurf2WTandSmurf2C716AchallengegrewsimilarlytoAHCYL1
knockdownwithlenti‐viruscontrolvector,allthreegrewsimilarlytonon‐silencing
knockdownwithSmurf2WTandSmurf2C716Achallenge.
CDC42BPA
CDC42BPAisknowntoregulatep53isthesenescencepathway(Sirotkinet
al.,2008).CDC42BPA(CDC42bindingproteinkinasealpha)knockdownisshownto
inhibittheexpressionofp53andPCNA,orproliferatingcellnuclearantigenin
ovariancells(Sirotkinetal.,2008).PCNApromotesDNAsynthesisduringSphase
andprovidesprotectionfromapoptosisduringG1phase(Sirotkinetal.,2008).
MyresultsindicatethatknockdownofCDC42BPAisnotsufficienttoallow
fibroblaststoescapeNotch3‐inducedsenescence;however,theknockdownwas
sufficienttopartiallyrescuecellsfromSmurf2‐inducedsenescence.Itispossible
thattheknockdownlevelofCDC42BPAinmyexperiments,whilesufficientto
interferewithitsfunctionwhiledownstreamofSmurf2,wasnotlowenoughto
37
interferewiththefunctionofCDC42BPAwhendownstreamofNotch3.Because
CDC42BPAisonlyshowntointeractwithoneofthesenescencepathways,thismay
notbesufficientforescapeofNotch3‐inducedsenescence.Itwillbeinterestingto
furtherdeterminetheinteractionofSmurf2withCDC42BPA,aswellasthe
interactionofCDC42BPAwithothergenesinthesenescencepathway.Thisresearch
maybeabletoenlightenusonifandhowCDC42BPAinteractswithNotch3.
Smurf2InVivo
Smurf2overexpressionwasabletoaffectgrowthinyoungmiceforashort
periodoftime.However,themiceseemedtohavecompensatedforthisabnormal
development,asthisstuntedgrowthlastedforlessthantwomonths.Smurf2
overexpressioninyoungmiceseemedtoadheretoasimilarsenescencepathway
thatoccursinvitro.Smurf2wasshowntoregulatep21,whichisconsistentwithin
vitrodata.However,asp16didnotseemtoberegulatedbySmurf2,thiscouldhave
occurredduetotheyoungageofthemice.p16hasbeenseentobevastlyincreased
duringsenescence(Alcorta,etal.,1996),indicatingthatp16willmostlikelynot
becomepresentinmiceuntilanolderage.
GrowthratesofSmurf2overexpressedmiceshouldberepeated,possibly
withweightsbeingmeasuredatmorefrequentintervals.Geneexpressionshould
alsobedeterminedatadditionalagestodetermineiftheexpressionchangesasthe
miceage.OurlabalsocontainsSmurf2C716Amice,itwillbeinterestingtoseeif
overexpressionofSmurf2C716AgivessimilarconsequencesasSmurf2WT
overexpression.
38
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