SupplementaryMaterialfor “Roleofp38MAPKandoxidativestressincopper-inducedsenescence” submittedin “Age”
EmmanuelleBoilan1,VirginieWinant1,EliseDumortier1,Jean-PascalPiret1,FrançoisBonfitto1,HeinzD.Osiewacz2,FlorenceDebacq-Chainiaux1andOlivierToussaint1*
1NARILISURBC,UniversityofNamur(FUNDP),Namur,Belgium,
2InstituteofMolecularBiosciences,JohannWolfgangGoetheUniversity,FrankfurtamMain,Germany
*correspondingauthor
DrO.Toussaint,
NARILISURBC,UniversityofNamur(FUNDP)
61,ruedeBruxelles,
B-5000Namur,Belgium
Phone:+3281724132
Fax:+3281724135
e-mail:
Methods
CellcultureandexposuretoCuSO4 - see the article
Extractionofproteins - see the article
Westernblotanalysis - see the article
MTTassay
WI-38HDFswereseededat14,000cells/cm2.Cellsurvivalwasassessedat0or24haftertheendoftheincubationwithCuSO4usingthe3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium(Sigma,USA)(MTTmethod)(Mosmann1983).CellswerewashedwithPBSandincubatedwithMTT+completeBME(BME+10%FBS)(1:1)for4hin5%CO2at37°C.Thesupernatantwasremovedandlysisbuffer(30%SDSandN,N-dimethylformamide,pH4,7,2:1)wasadded.Thecellswereincubatedat37°Cunderagitationfor1h.Theplatewasreadat570nmwiththeMicroplateReader(Bio-rad,microplatespectrophometer,USA).Triplicateswereperformed.
Statisticalanalysis
Statistical analysis was carried out with the Student's t-test. Ns, non significant (P > 0.05); *, 0.05 > P > 0.01; **, 0.01 > P > 0.001; ***, P < 0.001.
Results
DeterminationofsublethalconcentrationofCuSO4inWI-38HDFs
KnowingthatCuaccumulateswithRS,wewonderedwhetheraCuaccumulationcaninducecellsenescencewhenWI-38HDFsaretreatedwithsublethalconcentrationofcoppersalts.WetestedpreviouslydifferentconcentrationsofCuSO4orCuCl2toinduceaCuaccumulationinWI-38HDFsandNa2SO4orNaCltoverifytheimpactofsulfateorchlorideoncells.TreatmentwithCuinducesanincreaseofexpressionofcopper-regulatedgenes,suggestingamodificationofCuhomeostasisincells.TheseresultsweresimilarforthetwoCusaltsandnoeffectofsodiumsaltswasdetected(Scheckhuberetal.2009).WeusedCuSO4forthefollowingexperiments.WetreatedWI-38HDFswithCuSO4atconcentrationsupto1,000µMfor16h.Fig. S1aSupplementarymaterialdisplayspicturesinopticalmicroscopyjustattheendandat24hafterendofCuSO4treatmentfor16h.Thetreatmentswith750and1,000µMCuSO4for16hwereclearlycytotoxicjustattheendandat24haftertheendoftreatment.
AnalysisofcellviabilitywasperformedwiththeMTTmethod.Fig. S1bSupplementarymaterialrepresentstheviabilityforeachconcentrationcomparedtoviabilityjustbeforethetreatmentwithCuSO4(100%±10.4)toconsidertheimpactofbothcelldeathandproliferation.WhenmeasuredjustattheendoftreatmentwithCuSO4(1),viabilityofcellstreatedwith0(CTL),250and500µMCuSO4wasnotstatisticallydifferentfromtheresultsobtainedjustbeforethetreatmentwithCuSO4whereas750and1,000µMwerecytotoxic.
At24hafterendoftreatmentwithCuSO4(2),mostofcellstreatedwith750and1,000µMCuSO4weredead.Cellstreatedwith0(CTL)and250µMCuSO4proliferatedtothesameextent(around200%).At500µMCuSO4,wefoundonly127.4±12.6%,indicatinganeffectofCuSO4oncellproliferation.Thus,250and500µMofCuSO4wereconsideredassublethalconcentrations.Theconcentrationandtime-dependenteffectofCuSO4matchedverywellwithamathematicalmodelofcelldeathandinhibitionofcellproliferationcausedbyoxidantcompoundslikeH2O2,ethanolortert-butylhydroperoxide(Toussaintetal.1996).
LethalconcentrationsofCuSO4induceapoptosis
Usingwesternblotting,wedeterminedwhetherapoptosismarkerswereobservedjustattheendandat24haftertheendofthetreatmentwithCuSO4at250,500,750or1,000µMfor16h(Fig. S2Supplementarymaterial).WI-38HDFstreatedwithEtoposideat100µMfor16hwereusedaspositivecontrol.WeobservedtheincreaseforabundanceofPARP-1cleavedformanddegradationofitstotalform,andtheactiveformofcaspase3at24hafterendoftreatmentwithEtoposide.Theactiveformofcaspase3wasdetectedat750and1,000µMofCuSO4,attheendofthetreatmentand at24haftertheendofthetreatmentwithCuSO4.AbundanceofPARP-1cleavedformanddegradationofitstotalformwereincreasedat750and1,000µMCuSO4withtheclearesteffectat24haftertheendofthetreatmentwithCuSO4.Abundanceofdifferentcytoskeletonproteins,α-tubulin,β-actinandcofilin,wasdecreasedat750and1,000µMCuSO4suggestingthatapoptosishadtakenplace.Inthefollowingexperiments,wetreatedcellswith500µMCuSO4for16htoremaininsublethalconditions.
References
T.Mosmann, “Rapidcolorimetricassayforcellulargrowthandsurvival:applicationtoproliferationandcytotoxicityassays,” JournalofImmunologicalMethods,vol.65,no.1-2,pp.55-63,1983.
C.Q.Scheckhuberetal., “Age-relatedcellularcopperdynamicsinthefungalageingmodelPodosporaanserinaandinageinghumanfibroblasts,” PloSOne,vol.4,no.3,p.e4919,2009.
O.Toussaint,D.Lambert,J.Remacle, “Mathematicalmodelofthesurvivalcurvesandoftherecoverytimesformitosisofcellpopulationsexposedtostresses,” Invitrotoxicology,9(1996)251-259.
FigureLegends
Fig. S1 SupplementarymaterialDeterminationofsublethalconcentrationofCuSO4inWI-38HDFs.
(a)Imagesofcellsjustattheendand24haftertheendoftreatmentwith0(CTL),250,500,750or1,000μMCuSO4for16husinganopticalmicroscopewithphasecontrast.Magn.:63x.(b)MTTassayjustattheend(1)and24hafter(2)theendofthetreatmentwith0(CTL),250,500,750or1,000μMCuSO4for16h.Theresultsareexpressedinpercentagecomparedtocellularsurvivaljustbeforetreatment(100%)asmeans±S.D.from3independentexperiments.Statisticalanalysis:Student'st-test:ns,nonsignificant(P0.05);*,0.05P0.01;**,0.01P0.001.
Fig. S2 SupplementarymaterialLethalconcentrationsofCuSO4induceapoptosis.
Western-blotanalysisofPARP-1,α-tubulin,β-actin,cofilinandactivatedcaspase-3(cleaved-form)justattheendand24haftertheendofthetreatmentwith0(CTL),250,500,750or1,000μMCuSO4for16h. β-actinproteinwasusedascontrolloading.WI-38HDFstreatedwith150μMofEtoposidefor16hwasusedaspositivecontrol.
Fig. S3 SupplementarymaterialQuantification of western-blot analysis presented at Fig. 5
Quantification of the bands obtained by western-blotting was performed by the specific Odyssey program for quantification. We used the values for α-tubulin or lamin-B to evaluate the variations between samplesand we report all values on the control conditions (CTL = 1). The results are expressed under induction-fold of CTL.
Fig. S4 SupplementarymaterialQuantification of western-blot analysis presented at Fig. 6
Quantification of the bands obtained by western-blotting was performed by the specific Odyssey program for quantification. For p38MAPK, we calculated ratios between phospho-p38MAPK and p38MAPK.We used the values for α-tubulin or lamin-B to evaluate the variations between samplesand we report all values on the control conditions (CTL = 1). The results are expressed under induction-fold of CTL.
Fig. S5 Supplementary material SB 203580 inhibitory effect on phosphorylation of MAPKAPK-2 and p38MAPK
Western-blot analysis of phospho-MAPKAPK-2 and phospho-p38MAPK just at the end of the treatment with 500 μMCuSO4for16h or not, 20 μM SB 203580 for 16 h or not. Positive control was performed with 25 μM interleukin-1 (IL-1) for 1h or not, 20 μM SB 203580 for 1 h or not. β-actin was used as control loading.
Tables:Table S1: ListofPCRamplificationprimers,Table S2:Listofantibodies