Description:
NRF2/AREResponsiveLuciferaseReporterMCF7StableCellLineisderivedfromhumanbreastcancer,andstablyexpressfireflyluciferasereportergeneunderthecontroloftheNRF2/AREresponseelement. ThiscelllineisanidealcellularmodelformonitoringtheactivationofAntioxidantReceptorSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
NRF2isatranscriptionfactor,whichplaysanimportantroleinrespondingtooxidativestress.Undernormalcellularconditions,NRF2formsaproteincomplexwithKeap1inthecytoplasm,whichresultsintheproteasomaldegradationofNRF2causingittobeinactive. OxidativestressleadstotheactivationofanumberofkinasesincludingMAPK,ERK,p38,PKC,andPI3K. TheyphosphorylatebothKeap1andNRF2,whichdisrupttheKeap1-NRF2complex,andstimulatethetranslocationofNRF2tothenucleus,whereitformsacomplexwithMafproteins. TheNRF2/Mafheterodimersbinddirectlytoantioxidantresponseelements(AREs)locatedwithinpromotersofNRF2targetgenesandcoordinatetheexpressionofantioxidantgeneproducts.
SignosishasdevelopedaNRF2-reporterMCF7stablecelllinethathasbeen stablytransfectedwithpTA-NRF2-luciferasereportervector,whichcontains8repeatsofNRF2bindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion,alongwithaG418expressionvector. Thiscelllinecanbeusedtoinvestigateoxidativestress-mediatedactivationofupstreamkinasesandtoscreenanticancerdrugsthatcaninduceARE-drivengeneexpression.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisoftheNRF2PathwayReporterMCF7StableCellLineinresponsetostimuli. Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenweretreatedwithorwithout50nMTBHQrespectivelyinDMEMand0.1%FBSfor16hours.
