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Medchemexpress/Forskolin(Synonyms: Coleonol; Colforsin)/HY-15371/10mM*1mL in DMSO
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Medchemexpress/Forskolin(Synonyms: Coleonol; Colforsin)/HY-15371/10mM*1mL in DMSO
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MedChemExpress
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HY-15371-10mM*1mLinDMSO
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Forskolinisapotentadenylatecyclaseactivator,withbinding(IC50=41nM)toandactivation(EC50=0.5μM)oftypeIadenylylcyclase.
Description

Forskolinisapotentadenylatecyclaseactivator,withbinding(IC50=41nM)toandactivation(EC50=0.5μM)oftypeIadenylylcyclase.

IC50&Target

IC50:41nM(Adenylylcyclase)[1]
EC50:0.5μM(Adenylylcyclase)[1]

InVitro

Forskolin(Fsk)isanaturallyoccurringditerpeneisolatedfromColeusforskholii,directlyactivatesadenylylcyclase(AC)throughitscatalyticsubunittoincreaseintracellularlevelsofcyclicadenosinemonophosphate(cAMP)[1].Forskolin(Fsk)affectstheproliferationofthehumanT-celllinessuchasKit225andMT-2.ForskolintreatmentinhibitstheproliferationofbothKit225andMT-2cellsinadose-dependentmannerwithanIC50equalto~5μMFsk.Forskolintreatment(10-100μM)increasescAMPilevels~5-to20-foldabovebasallevels,whichreachemaximumlevelsbetween50-100μMForskolin[2].

InVivo

TheForskolin(Fsk)-treatedMrp4-/-miceshowsanincreasednumberofKi67-positiveandcleavedcaspase3-positiveECs,asignificantdecreaseintheamountofpericytecoverage,andareducednumberofemptysleeves.Inpupsexposedtohyperoxia(75%oxygen)fromP7toP12,theMrp4-/-miceshowsasignificantincreaseintheunvascularizedretinalarea[3].Theaveragebloodglucoseinthehealthyratgroupis102.12±1.94mg/dL,101.25±3.56forcontrolgroupand103±2.08inforskolingroup.Thedatashowsthatglucoselevelsattheendofthestudyarelowerinforskolingroup,withasignificantdifferenceaccordingtothestatisticaltestsapplied(p=0.03)[4].

References

  • [1].RobbinsJD,etal.Forskolincarbamates:bindingandactivationstudieswithtypeIadenylylcyclase.JMedChem.1996Jul5;39(14):2745-52.

    [2].RodriguezG,etal.Forskolin-inducIBLecAMPpathwaynegativelyregulatesT-cellproliferationbyuncouplingtheinterleukin-2receptorcomplex.JBiolChem.2013Mar8;288(10):7137-46.

    [3].MatsumiyaW,etal.ForskolinmodifiesretinalvasculardevelopmentinMrp4-knockoutmice.InvestOphthalmolVisSci.2012Dec7;53(13):8029-35.

    [4].Ríos-SilvaM,etal.EffectofchronicadmiNISTrationofforskolinonglycemiaandoxidativestressinratswithandwithoutexperimentaldiabetes.IntJMedSci.2014Mar11;11(5):448-52.

PreparingStockSolutions
ConcentrationVolumeMass1mg5mg10mg
1mM2.4361mL12.1803mL24.3605mL
5mM0.4872mL2.4361mL4.8721mL
10mM0.2436mL1.2180mL2.4361mL
Pleaserefertothesolubilityinformationtoselecttheappropriatesolvent.
KinaseAssay
[2]

ForJak3kinaseassays,Fsk-treatedMT-2cellsarelysed,clarified,andimmunoprecipitatedusingJak3antibody.Kinasereactionsarecarriedoutat30°Cfor20min.ForPKAkinaseassays,untreatedMT-2cellsarelysed,andJak3isimmunoprecipitatedandboundtoPASbeads.ImmunoprecipitatedJak3iswashedwithkinasebuffer(50mMHepes-NaOH(pH7.4),10mMMgCl2,0.5mMEGTA,0.5mMDTT,20μg/mLaprotinin,10μg/mLleupeptin,1μg/mLpepstatinA)andincubatedwith200μMATPandpurifiedproteinkinaseAcatalyticsubunit(PKAc)asindicatedinthefigurelegends.Kinasereactionsarecarriedoutat32°Cfor30minfollowedbyvigorouswashingofthebeadswithcoldkinasewashbuffer.For[γ-32P]ATPrADIolabeledkinaseassaysusingrecombinantJak3,Hek293cellsaretransfectedwithwildtype(WT)Jak3orkinase-deadJak3K855AusingLipofectamine2000accordingtothemanufacturer"sinstructions.CellsarelysedandimmunoprecipitatedwithJak3antibody.Jak3-boundPASbeadsarewashedthreetimesincoldlysisbufferfollowedbykinasebuffer.Kinasereactionsareinitiatedbyadding10μCi[γ-32P]ATP,10μMunlabeledATP,and1μgofpurifiedPKActoJak3-boundPASbeadreactionmixtures.Kinasereactionsareperformedat32°Cfor30min.Jak3-boundPASbeadsarewashedthreetimesinradioimmunoassaybuffer(10mMTris-HCl,pH7.4,75mMNaCl,20mMEDTA,10mMEGTA,20mMNa4P2O7,50mMNaF,20mM2-glycerolphosphate,1mMp-nitrophenylphosphate,0.1%TritonX-100)andonetimeinkinasewashbuffer.Thereactionsarestoppedbyadding2×SDS-PAGEsamplebufferfollowedbySDS-PAGE.CoomassiestainableJak3bandsareexcisedfromthePVDFmembraneandsubjectedtophosphoaminoacidanalysis[2].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

CellAssay
[2]

Forskolin(Fsk)isdissolvedinDMSOandstored,andthendilutedwithappropriatemedia(DMSO1%)beforeuse[2].

QuiescentKit225orMT-2cellsareseededinto96-wellplatesat5×104cellsperwell.Cellsarethenpretreatedfor1hwith1%DMSO(vehicle)orForskolinat1,5,10,25,50,and100μMconcentrations.ThecellsarestimulatedwithIL-2andculturedforanadditional20hat37°C.Controlcellsaretreatedwith1%DMSOfor20h.Duringthefinal4hofincubation,thecellsarepulsedwith[3H]thymidineataconcentrationof0.5μCi/200μL.Cellsareharvestedontofiberglassfiltersandanalyzedusingliquidscintillationcounting[2].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

AnimalAdministration
[3][4]

ForskolinisdissolvedinDMSOandthendiluted(Mice)[3].

Mice[3]
C57BL/6Jmiceareused.Mrp4-knockoutmice,whichareestablishedandrepeatedlybackcrossedtotheC57BL/6Jmice.Forskolinisinjectedintraperitoneallyintoneonatalmiceatpostnataldays4(P4)and5(P5).MiceinjectedwithDMSOserveasthecontrols.ThetreatedmiceareeuthanizedatP6,andtheirretinasareisolatedforwhole-mountimmunohistochemistry(IHC).TheeffectofdifferentconcentrationsofForskolinonthesurvivalrateandretinalvasculatureisfirsttested,andtheoptimalconcentrationisdetermined,1.0μg/50μL(0.3mg/kg)atP4and1.5μg/50μL(0.5mg/kg)atP5,usedtocomparetheretinalvascularphenotypesbetweenWTmiceandMrp4-deficientmice.
Rat[4]
MaleWistarrats,aged10-14weeksold,withameanweightof300g±50g,aredividedintofourgroups;19areexperimentallyinducedtodevelopdiabetes,and8aremaintainedinahealthycondition.BothdiabeticandhealthyratsreceivenoForskolin(control),or6mg/kgperdayofForskolin,administeredorallyfor8weeks.BloodglucoselevelsaredeterminedineachgroupbeforeandafterForskolintreatment.Thediabeticratsaretestedtwoweeksafterconfirmingthepresenceofdiabetes(threeweeksaftertheinduction)andaftereightweeksofthedesignatedtreatment.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

References

  • [1].RobbinsJD,etal.Forskolincarbamates:bindingandactivationstudieswithtypeIadenylylcyclase.JMedChem.1996Jul5;39(14):2745-52.

    [2].RodriguezG,etal.Forskolin-induciblecAMPpathwaynegativelyregulatesT-cellproliferationbyuncouplingtheinterleukin-2receptorcomplex.JBiolChem.2013Mar8;288(10):7137-46.

    [3].MatsumiyaW,etal.ForskolinmodifiesretinalvasculardevelopmentinMrp4-knockoutmice.InvestOphthalmolVisSci.2012Dec7;53(13):8029-35.

    [4].Ríos-SilvaM,etal.Effectofchronicadministrationofforskolinonglycemiaandoxidativestressinratswithandwithoutexperimentaldiabetes.IntJMedSci.2014Mar11;11(5):448-52.

MolecularWeight

410.5

Formula

C₂₂H₃₄O₇

CASNo.

66575-29-9

Storage
Powder-20°C3years
 4°C2years
Insolvent-80°C6months
 -20°C1month
Shipping

RoomtemperatureincontinentalUS;mayvaryelsewhere

Solvent&Solubility

DMSO:≥32mg/mL

*"<1 mg/ml"="" means="" slightly="" soluble="" or="" insoluble.="" "≥"="" means="" soluble,="" but="" saturation="">

Purity:98.60%

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