Description:
NRF2/AREResponsiveLuciferaseReporterHepG2 CellLineisderivedfromHumanLivercancer,andstablyexpressfireflyluciferasereportergeneunderthecontrolofNRF2/AREresponseelement.ThiscelllineisanidealcellularmodelformonitoringtheactivationofAntioxidantresponse Pathway triggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
NRF2playsacrucialroleincellularanti-oxidantdefense,makingitatherapeutictargetforneurodegenerativediseasesandcancer. Undernormalconditions,NRF2localizesinthecytosolandisrapidlydegradedbytheproteasome. Underoxidativestress,NRF2isstABIlizedandtranslocatestothenucleuswhereitbindstoaDNApromoterandinitiatesgeneexpression. Inthenucleus,NRF2formsaheterodimerwithasmallMafproteinandbindstotheAntioxidantResponseElementintheupstreampromoterregionofmanyantioxidativegenes,andinitiatestheirtranscription.
ThisNRF2luciferasereporterstablecelllinehasbeenstablytransfectedwithpTA-ARE-luciferasereportervector,whichcontains4repeatsofantioxidantresponsebindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion,alongwitha hygromycinexpressionvector. Followingselection,the hygromycinresistantclonesweresubsequentlyscreenedforTBHQ-inducedluciferaseactivity.Theclonewiththehighestfoldinductionwasselectedandexpandedtoproducethisstablecellline.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisoftheNRF2PathwayReporterHePG2StableCellLineinresponsetostimuli. Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.ThecellsthenweretreatedwithoutorwithdifferentconcentrationTBHQrespectivelyinDMEMand0.1%FBSfor16hours.
