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CancerResearchApplicationNoteNo.10June2011CancerResearchIntroductionWorkflow:AnalysisofThep53tumorsuppressorplaysacentralroleinthecellulardefenseagainstcancer.Itcontrolsthep53pathway(acomplexNutlin-InducedChangessignalingnetwork),anditcanstopcellproliferationandinduceapoptosisinresponsetodiverseoncogenicstresses.inmRNASynthesisandThecellularlevelofp53istightlycontrolledbyMDM2,aspecificE3ubiquitinligase.MDM2bindsp53initsProteinExpressionusingN-terminalregionandblocksthetranscriptionalactivityofp53,butthecellularlevelofMDM2dependsonthethexCELLigenceSystem,transcriptionalactivityofp53.MDM2andp53mutuallyregulatetheircellularlevelsthroughanegativefeedbackqRT-PCR,NorthernBlots,loop.Wild-typep53representsanattractivetargetforpharmacologicalintervention.OneofthemainapproachesandWesternBlotsistoactivateitincancercellsbyinhibitingMDM2tobindtop53.Recently,small-moleculeinhibitorsofthep53/MDM2interaction,thenutlins,havebeenidentified.Theyrepresentvaluabletoolsforstudyingp53regulationandleadReginaWeil,ClaudiaVorwerkandBarbaraRuegertothedevelopmentofcancertherapeutics(Carvajaletal,Tovaretal).Forlifescienceresearchonly.Notforuseindiagnosticprocedures.IntroductioncontinuedInthisstudy,wedescribeamodelsystemofthecellularoverexpressionofMDM2(Vassilevetal,Tovaretal).effectsofnutlin3a(activeenantiomer)andnutlin3bExponentiallygrowingSJSA1cellsweretreatedeitherwith(inactiveenantiomer)usingcontinuouscellrecordingwithnutlin3a,nutlin3b,orDMSO(nutlinsolvent),andcellthexCELLigenceSystem,anddetailedanalysisofmRNAgrowthwasmonitoredusingthexCELLigenceSystem.Timesynthesisandproteinexpressionusingnorthernandpointsat2,6,and24hoursforRNAcollectionandproteinwesternblots.ThisworkflowisapowerfuladditionalmodelextractpreparationwereselectedaccordingtotheCellIndexsystemforcellularanalysis,aftertheoptimaltimepoints(CI)profilesrecordedusingthexCELLigenceSystemforfunctionalendpointassayshavebeendefinedbythe(seeCancerResearchApplicationNote1).Subsequently,xCELLigenceSyste
