Description:
SMAD2/3ResponsiveLuciferaseReporterNIH/3T3StableCellLineisderivedfrommousefibroblast,andstablyexpressfireflyluciferasereportergeneunderthecontrolofSMAD2/3responseelement. Thiscelllineisanidealcellularmodelformonitoringtheactivation ofTGFbeta SignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
Smadproteinsaretranscriptionfactorsthatrespondtotransforminggrowthfactor-β(TGFβ)signaling,whereTGFβinducesitsmembranereceptorstodirectlyactivateSmadproteins. TheseactivatedSmadscomplexwithSmad4(co-Smad),translocatefromcytoplasmintonucleusandbindtotargetpromoterregiontoregulategenetranscriptions. DysfunctioninTGFβpathwayleadstoimmunosuppressionandangiogenesis,whichcanmakecancermoreinvasive.
SignosishasdevelopedSMAD/TGFbluciferasereporterstablecelllinebyco-transfectingSMADluciferasereportervectorandhygromycinexpressionvector. ThehygromycinresistantclonesweresubsequentlyscreenedforTGFb1-inducedluciferaseactivity. ThecelllinecanbeusedasareportersystemformonitoringtheactivationofSMADtriggeredbystimulitreatment,suchasTGFb1,andgeneoverexpressionandgeneknockdown.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofSMAD/TGFbetaLuciferaseReporterNIH/3T3StableCellLine.NIH/3T3cellsstablyexpressingSMADluciferasereporterweretreatedwith20ng/mLTGF-beta1inDMEM+0.1%FBSfor16hours. TGF-betaisoformsinducedabout8-foldincreaseinSMAD-luciferasereporteractivity.
