Signosis/NFAT Luciferase Reporter Hela Stable Cell Line/SL0018NP/1 Ea

Description:

NFATResponsiveLuciferaseReporterHealStableCellLine isderivedfromhumancervicalcancer,andstablyexpressfireflyluciferasereportergeneunderthecontrolofNFATresponseelement. Thiscelllineisanidealcellularmodelformonitoringtheactivation  ofCalciumSignalingReceptorSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.

Principle:

NFATsareafamilyoftranscriptionalfactorsthatplayanimportantroleinimmuneresponseaswellasinthedevelopmentofcardiac,skeletalmuscle,andnervoussystems. NFATsareregulatedbycalciumsignaling.Throughcalciumactivationofthephosphatasecalcineurin,NFATcproteinstranslocatefromthecytoplasmintothenucleus,wheretheycooperatewithotherproteinstomediategeneexpression.NuclearimportofNFATisblockedbykinasessuchasPKAandGSK3.NFATsarealsoimplicatedinbreastcancer. SignosishasestablishedaNFATluciferasereportercelllinethathasbeenstablytransfectedwithaNFAT-luciferasereporterconstruct.Viatheanalysisofluciferase,thecelllinecanbeemployedtomonitorthecellularchangesofNFATactivitiesthataretriggeredbystimulation,compoundtreatment,enforcedgeneexpressionorgeneknockdown. 

Thecelllineisestablishedbytransfectionusinga pTA-NFAT-luciferasereportervector,whichcontainsNFATbindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion, alongwithhygromycinexpressionvectorfollowedbyhygromycinselection. ThehygromycinresistantclonesweresubsequentlyscreenedforPMA+ionomycin-inducedluciferaseactivity.

 

PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation.

 

Data:

 

 

 

 

 

 

 

 

 

 

AnalysisofNFATLuciferaseReporterHelaStableCellLine.  Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenweretreatedwithorwithout10ng/mlPMAand1uMionomycininDMEMand0.1%FBSfor6hours. Morethan30foldincreaseinluciferaseactivitywasdetectedwhencomparedtountreatedcells.