Description:
SMAD2/3ResponsiveLuciferaseReporterHepG2StableCellLineisderivedfromhumanlivercancer,andstablyexpressfireflyluciferasereportergeneunderthecontroloftheSMAD2/3responseelement.Thiscelllineisanidealcellularmodelformonitoringtheactivation ofTGFbetapathwayReceptorSignalingPathwaytriggeredbystimulitreatment,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/TGFbetaLuciferaseReporterHepG2StableCellLine.HepG2cellsstablyexpressingSMADluciferasereporterweretreatedwithdifferentconcentrationof TGF-beta1 inEMEM+0.1%FBSfor18hours.
