NARINGENIN IMPAIRS TWO PORE CHANNEL-2 …10.1038/s41598-017... · NARINGENIN IMPAIRS TWO PORE...
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NARINGENIN IMPAIRS TWO PORE CHANNEL-2 ACTIVITY AND INHIBITS VEGF-INDUCED ANGIOGENESIS Irene Pafumi, Margherita Festa, Francesca Papacci, Laura Lagostena, Cris8na Giunta, Vijay Gutla, Laura Cornara, Annarita Favia, Fiore@a Palombi, Franco Gambale, Antonio Filippini and Armando Carpaneto Suppl. Fig. 1: the human TPC1 channel is expressed in TPC-free Arabidopsis vacuoles (a) Currents recorded in the absence (leE) and in the presence (right) of 90 nM PI(3,5)P 2 added in the cytosolic bath solu8on. Currents were elicited by voltages ranging to -90 to +80 mV in 10 mV step. Holding and tail voltages were respec8vely of -70 and -50 mV. (b) Steady-state currents obtained as the mean values of the last 100 ms recording were plo@ed against the applied voltages. Empty and filled symbols indicated the absence and the presence of 90 nM PI(3,5)P2. Data, shown as mean± s.e.m., were from 7 different vacuoles. Suppl. Fig. 1
Transcript of NARINGENIN IMPAIRS TWO PORE CHANNEL-2 …10.1038/s41598-017... · NARINGENIN IMPAIRS TWO PORE...
NARINGENINIMPAIRSTWOPORECHANNEL-2ACTIVITYANDINHIBITSVEGF-INDUCEDANGIOGENESISIrenePafumi,MargheritaFesta,FrancescaPapacci,LauraLagostena,Cris8naGiunta,VijayGutla,LauraCornara,AnnaritaFavia,Fiore@aPalombi,FrancoGambale,AntonioFilippiniandArmandoCarpaneto
Suppl.Fig.1:thehumanTPC1channelisexpressedinTPC-freeArabidopsisvacuoles(a) Currentsrecordedintheabsence(leE)andinthepresence(right)of90nMPI(3,5)P2addedinthecytosolicbathsolu8on.Currentswereelicitedbyvoltagesrangingto-90to+80mVin10mVstep.Holdingandtailvoltageswererespec8velyof-70and-50mV.(b)Steady-statecurrentsobtainedasthemeanvaluesofthelast100msrecordingwereplo@edagainsttheappliedvoltages.Emptyandfilledsymbolsindicatedtheabsenceandthepresenceof90nMPI(3,5)P2.Data,shownasmean±s.e.m.,werefrom7differentvacuoles.
Suppl.Fig.1
AtTPC1
NARINGENINIMPAIRSTWOPORECHANNEL-2ACTIVITYANDINHIBITSVEGF-INDUCEDANGIOGENESISIrenePafumi,MargheritaFesta,FrancescaPapacci,LauraLagostena,Cris8naGiunta,VijayGutla,LauraCornara,AnnaritaFavia,Fiore@aPalombi,FrancoGambale,AntonioFilippiniandArmandoCarpaneto
Suppl.Fig.2:NaringenininhibitstheArabidopsisthalianaTPC1AtTPC1channelresponsetovoltages8mula8onatV=+80mVincontrol,inthepresenceof300µMcytosolicNarandinrecoverycondi8onsinvacuolesfrommesophyllcellsofArabidopsisthalianaplants.Ionicsolu8ons:200mMKCl,2mMMgCl2,2mMCaCl2,10mMMES/Tris,pH5.5inthepipe@e;100mMKCl,2mMMgCl2,1mMCaCl2,1mM dithiothreitol (DTT),and10mMHEPES/Tris,pH7.5 in thebath;osmolarityadjusted to600mOsmby theaddi8onofD-sorbitol.Similarexperimentswereperformedfromatleast15differentvacuoles.
Suppl.Fig.2
pVEGFR2
α-tubulin
VEGF
a b
NARINGENINIMPAIRSTWOPORECHANNEL-2ACTIVITYANDINHIBITSVEGF-INDUCEDANGIOGENESISIrenePafumi,MargheritaFesta,FrancescaPapacci,LauraLagostena,Cris8naGiunta,VijayGutla,LauraCornara,AnnaritaFavia,Fiore@aPalombi,FrancoGambale,AntonioFilippiniandArmandoCarpaneto
Suppl.Fig.3:PhosphorylaRonofVEGFR2isnotaffectedbynaringenintreatment.Phosphoryla8onstateofVEGFR2atTyr1175,evaluatedbywesternblotinuntreated(NT)orVEGF-treatedHUVECs.CellswerepreincubatedwithNar(200µM,600µMand1000µM)orvehiclefor30min,orwithTSU-68(2,1µM),oritscontrol(vehicle-2)for1h.Sampleswerethens8mulatedwith100ng/mlVEGFfor15min(a).TheintensityofpVEGFR2bandswasquan8fiedandnormalizedtoα-tubulincontent.Datainbarchartrepresentmean±s.e.m.fromthreeindependentexperiments(b).Asapparent,VEGF-inducedreceptorphosphoryla8onissignificantlyinhibitedbyTSU-68butnotbyNar.
Suppl.Fig.3
a b
NARINGENINIMPAIRSTWOPORECHANNEL-2ACTIVITYANDINHIBITSVEGF-INDUCEDANGIOGENESISIrenePafumi,MargheritaFesta,FrancescaPapacci,LauraLagostena,Cris8naGiunta,VijayGutla,LauraCornara,AnnaritaFavia,Fiore@aPalombi,FrancoGambale,AntonioFilippiniandArmandoCarpaneto
Suppl.Fig.4:Neither[Ca+2]iincreasenortheformaRonofcapillary-liketubessRmulatedbyAng-1areaffectedbyNed-19.(a,b)Cellswerepretreatedfor30minwith100µMNed-19and s8mulatedwith100ng/mlAng-1.(a)Barchartshowingmaximum[Ca+2]i,***P<0.001;(b)CellswereplatedinMatrigel-coateddishesandincubatedfor2-3hinEGM-2supplementedwithAng-1orAng-1+Ned-19.Quan8ta8veevalua8onoftube forma8onas thenumberof closedpolygons formed in9fields for eachexperimental condi8on.Data inbar charts representpercentageof thecontrol(mean±s.e.m.ofthreeindependentexperiments).
Suppl.Fig.4
Vehicle VEGF Nar1000μM+VEGF Nar1000μMab
Suppl.Fig.5
Suppl.Fig.5:NaringeninimpairsVEGF-inducedcapillarytubeformaRoninvitro.(a)Representa8veimagesofoneofthreeindependentexperiments.HUVECswereplatedinMatrigel-coateddishesandincubatedfor4hinEBM-2+2%FBSsupplementedornotwithVEGForNar,orinmediumcontainingbothVEGFandNar.Eachcondi8onwastestedintriplicateforeachindividualexperiment.(b)Quan8ta8veevalua8onoftubeforma8onasthenumberofclosedpolygonsformedin6fieldsforeachexperimentalcondi8on.Datainbarchartrepresentmean±s.e.m.ofthreeindependentexperiment.*P<0.05.
NARINGENINIMPAIRSTWOPORECHANNEL-2ACTIVITYANDINHIBITSVEGF-INDUCEDANGIOGENESISIrenePafumi,MargheritaFesta,FrancescaPapacci,LauraLagostena,Cris8naGiunta,VijayGutla,LauraCornara,AnnaritaFavia,Fiore@aPalombi,FrancoGambale,AntonioFilippiniandArmandoCarpaneto
Suppl.Fig.6
Suppl.Fig.6:NaringenindoesnotimpairinvivovascularizaRoninducedbyAng-1In vivo vessel forma8on was assessed aEer subcutaneous injec8on of 5 weeks oldmale/female C57BL/6mice withMatrigel plugscontainingeithervehicleorAng-1(150ng/ml)orAng-1plus1000μMNar.FivedaysaEerinjec8onthemiceweresacrificedandplugvasculariza8onwasevaluatedas hemoglobincontentexpressedasabsorbance(OD)/1gmatrigelplug.HemoglobincontentinAng-1plugs(n=8)didnotsignificantlydifferfromthatinAng-1+Narplugs(n=8)(P>0.2).
NARINGENINIMPAIRSTWOPORECHANNEL-2ACTIVITYANDINHIBITSVEGF-INDUCEDANGIOGENESISIrenePafumi,MargheritaFesta,FrancescaPapacci,LauraLagostena,Cris8naGiunta,VijayGutla,LauraCornara,AnnaritaFavia,Fiore@aPalombi,FrancoGambale,AntonioFilippiniandArmandoCarpaneto
