CustomerValidation
- •JDermatolSci.2017Oct11.
- •BiochemBiophysResCommun.2016Dec2;481(1-2):90-96.
| Description | 3-MethyladenineisaselectivePI3KinhibitorwithIC50of25μMand60μMforVps34andPI3Kγ,respectively,anditcaninhibitallPtdIns3Ksat10mM. |
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| IC50&Target | IC50:25μM(Vps34),60μM(PI3Kγ) |
| InVitro | 3-methyladenineshowssomelimitedVps34preferenceinvitro,withanIC50of25µMforVps34ascomparedwith60µMforPtdIns3Kγ,and3-methyladenine(10mM)caninhibitallPtdIns3Ks[1].3-methyladenine(3-MA,5mM)suppressesautophagyinHeLacellsunderbothglucose-freeconditionsandnormalconditions.3-methyladenineinducescaspase-dependentcelldeathinHeLacells,andthedeathoccursindependentlyoftheinhibitionofautophagy.3-MA(1mM)significantlyshortensthedurationofnocodazole-induced-prometaphasearrest[2].Alargenumberofneuronsfromthe3-MAgroupareintheearlystageofapoptosis,displayingcellshrinkage,nuclearandchromatincondensationandaggregationattheedgeofthenucleus[3]. |
| InVivo | 3-methyladenine-treatmentsignificantlyaggravatesneurologicalsymptomsandapoptoticcelldeathafterSAH[3]. |
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Pleaserefertothesolubilityinformationtoselecttheappropriatesolvent. | ||||||||||||||||||||||||||||
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| CellAssay [2] | CellviABIlityisdeterminedbyatrypanblueexclusionassay.Cellsareculturedinthemediumwith3-Methyladenine.BothadherentandfloatingcellsarecollectedandsUSPendedinphosphatebufferedsaline(PBS,pH7.4)atafinaldensityof1-2×106/mL.Anequalvolumeof0.4%trypanbluesolution(w/v,inPBS)isaddedtothecellsuspensionandmixedthoroughly.Afterincubationatroomtemperaturefor3min,cellcountingisperformedusingahemacytometer.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly. AnimalAdmiNISTration | [3] 3-Methyladenineisdissolvedinnormalsaline. Animalsarepositionedinastereotacticframeandtreatedwith5-μLintracerebralventricularinjectionsofautophagyinducerRAP(20 pmol),inhibitor3-Methyladenine(3-MA,400 nmol)orvehicle20 minbeforeSAHonset.Intracerebralventricularinjectionsaremadeintotheright lateralventricle atcoordinatesof0.8 mmanteriortoposteriorbregma,1.5 mmmidtolateral,3.8 mmdorsaltoventral.Aftertheinjection,thesyringeisleftinthesitefor30 sbeforeremoval,andtheburrholeisfilledwathbonewax.Drugdosesareselectedonthebasisofpreviouspublishedstudiesandpilotexperimentsinourlaboratory.3-MAisdissolvedinnormalsalinebyheatingthesolutionto60-70°Cimmediatelybeforetreatment.RAPisfirstdissolvedinethanolandthendilutedwithnormalsaline(finalethanolconcentration<2%).=""mce=""has=""not=""independently=""confirmed=""the=""accuracy=""of=""these=""methods.=""they=""are=""for=""reference=""> References |
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