Description:
ATF6ResponsiveLuciferaseReporterCHO-K1StableCellLineisderivedfromChineseHamsterOvary,andstablyexpressfireflyluciferasereportergeneunderthecontrolofATF6responseelement. ThiscelllineisanidealcellularmodelformonitoringtheactivationofUnfoldedProteinResponse,ERstressSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
ATF6playsakeyroleintheUnfoldedProteinResponse,asATF6actssynergisticallywithotherERstresssensors,PERKandIRE1,tomitigateERstress. ATF6isactivatedbyproteolyticcleavageanduponactivation,ittranslocatestothenucleusandbindstocis-actingERstresselementthatispresentinpromotersofERchaperones.
SignosishasdevelopedATF6luciferasereporterstablecelllinebytransfectingcellswithplasmidscontainingATF6luciferasereporterandhygromycinexpressioncassette. Thehygromycinresistantclonesweresubsequentlyscreenedforthapsigarginortunicamycin-inducedluciferaseactivity. ThecelllinecanbeusedasareportersystemformonitoringtheactivityofATF6triggeredbystimulitreatment,geneoverexpressionandgeneknockdown.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofSL-0024ATF6reporteractivityinresponsetoERstress. TheCHO-K1cellswereseededona96-wellplateforovernightwithDMEM/F12including10%FBS.Thecellsthenweretreatedwithindicatedconcentrationsoftunicamycin(TM)orthapsigargin(TG)inDMEM/F12and10%FBSfor16hours. CHO-K1-ATF6LuciferaseReporterCellLineexhibitsstress-dependentincreaseinluciferaseactivitywhencomparedtocontrolcells.
