Description:
AP-1ResponsiveLuciferaseReporterHelaStableCellLineisderivedfromhumancervicalcancer,andstablyexpressfireflyluciferasereportergeneunderthecontrolofAP-1responseelement. Thiscelllineisanidealcellularmodelformonitoringtheactivation ofJNK,ERK,MAPKSignalingReceptorSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
AP-1playsakeyroleinthecontrolofcellproliferation,differentiation,transformation,survivalandapoptosis. cJunandFosdimerizetoformAP-1andthisproteincomplexselectivelybindstoDNAsequencescontainingtheTPA-responsiveelements(TREs)toregulategeneexpression. Manystimuli,bothphysiologicalandpathological,canregulateAP-1activity,includingTPA,serum,growthfactors,cytokines,stresssignals,infections,andoncoproteins,TNFandIL-1. AP-1activitycanbeusedasreadoutforJNK,ERK,orMAPKkinasesignalingpathway.
SignosishasdevelopedAP-1luciferasereporterstablecelllinebytransducingcellswithbaculoviruscontainingbothAP-1luciferasereporterandhygromycinexpressioncassette. ThehygromycinresistantclonesweresubsequentlyscreenedforPMA-inducedluciferaseactivity. ThecelllinecanbeusedasareportersystemformonitoringtheactivityofAP-1triggeredbystimulitreatment,suchasTNFa,IL-1,geneoverexpressionandgeneknockdown. Thecellscontainnoviralparticlesandrequirehandlingatbiosafetylevel1protocol.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofAP-1LuciferaseReporterHelaStableCellLine. Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenweretreatedwithorwithout10ng/mlPMAinDMEMand0.1%FBSfor6hours. Morethan50foldincreaseinluciferaseactivitywasdetectedwhencomparedtountreatedcells.
