WSP-1(H2Sprobe)wasobtainedfromShanghaiMaokangBiotechologyCo.,Ltd.ToverifytheintracellularROS-triggeredH2Srelease,acommerciallyavailableprobe(WSP-1),whosefluorescencecanbeselectivelyturnedonbyH2S,17wasusedforthedetectionofH2SinHeLacells.AsshowninFig.S17(imagesc–e),thefluorescenceofWSP-1inthegreenchannelriseswithincreasingthedoseofNABinthepresenceofH2O2,suggestingthegenerationofthegraduallyincreasedH2S.
Hydroxyphenylfluorescein(HPF)waspurchasedfromShanghaiMaokangBio.Co.(Shanghai,China).HPFwasusedtodetectthegenerationofOH.Typically,1mLBBNCsaqueoussolution(80μgmL-1)containingwithHPF(1μM)wereirradiatedby650nmlaser(0.5Wcm-2),thefluorescenceofHPFwasdetectedafter15min.HPFaqueoussolutionwithlaserservedascontrol.
Theaminophenylfluorescein(APF)astheHClOdetectorwerepurchasedfromShanghaiMaokangbio.ThefurtherEDTrelatedtherapeuticspeciesincludingtheROS,1O2andtheintermediateHClOhavebeendetectedintumourtissuesofdifferentgroupsbyusingthecorrespondingfluorescentprobes,DCFH-DA,SOSGandaminophenylfluorescein(APF,asHClOdetector).
FluorescentBrightener28wasobtainedfromMaokangBiotechnology(Shanghai,China).Forfluorescencestainingofextracellularpolymericsubstances(EPSs),afterthebiofilmformation,thePDMSsurfaceswereincubatedwith6μMSYTO9atroomtemperatureinthedarkfor12min,followedbyasecondstain-ingof0.15mMfluorescentBrightener28for3min.
Finally,withgentleagitation,themycelia(approximately0.2g)wereincubatedfor4–5hat31°Cin2mLof20mg/mLlywallzyme(ShanghaiMaokangBiologicalTechnologyCo,Ltd.,China,CatalogNo.MX7365-500MG)containing0.6Mmannitolforpreparingprotoplasts.Afterincubation,theseprotoplastswerewashedfreeofenzymemannitolsolutionandtransferredtoregenerationmedium(RM).
Cellswerepretreatedwith25ng/mlPMA(Sigma,StLouis,USA)for5hat37°C.Atthesametime,10μg/mlbrefeldin(MaokangBiotechnology,Shanghai,China)wasaddedintothecellsforthelast4hoftheincubation.
MX4510-100UGPBFIAM(K+Indicator)钾离子指示探针
Sodium-bindingbenzofuranisophthalateacetoxymethylester(SBFI-AM)andPotassium-bindingBenzofuranIsophthalateAcetoxymethylester(PBFI-AM)werepurchasedfromMKbio.China.
Bovineserumalbumin(BSA,StandardGrade)wasacquiredfromShanghaiMaokangBiotechnologyCo.,Ltd(China).Polyacrylonitrileultrafiltrationmembranes(PAN,MWCOrangingfrom10to300kDa)werepurchasedfromShanghaiMegaVisionMembraneEngineering&TechnologyCo.,Ltd(China).
Cellcountingkit-8reagentwaspurchasedfromShanghaiMaokangBiotechnologyCo.,Ltd.(China).CellproliferationonthescaffoldwasmeasuredwithCCK-8after1,3,5and7daysofculture,andcellcultureplateswereusedasblankgroup.TheBMSCs-seededhydrogelswereincubatedinDMEMcontaining10%CCK-8protectedfromlightatanincubatorof37°Cand5%CO2for4h,andmeasuredat450nmusingamicroplatereader.
Aftertheformazanwasdissolvedbydimethylsulfoxide(DMSO),theopticaldensity(OD)wasthendeterminedusingenzyme-labeledinstrument(iMark,BIO-RAD,US)atthewavelengthof570nm.TorevealtheviabilityofadherentMC3T3-E1cells,aLive/DeadDoubleStainingKit(MKBio,China).
HEK293cellswerefirsttransfectedwithpCAGGSandpCAGGS-PRDX1.WhenPRDX1washighlyexpressedforabout24h,cellswereinfectedwithDTMUVandtreatedfor6h,whereastheRosuppositivecontrolgroupwassetinparallel.Finally,theprobeofDCFH-DAwasdilutedat1:1000andtreatedat30°Cfor20minreferringtotheROSAssayKit(MKBio,Shanghai,China).Cellswerethenobservedunderafluorescentinvertedmicroscopeimmediately.
AAVparticleswerereleasedfromcellswiththreefreeze/thawcycles,followedbyincubationwith50U/mlbenzonasenuclease(MKbio)and10U/mlRNaseIat37°Cfor30min,and0.5%sodiumdeoxycholate(Sigma)treatmentforanother30min.
Ionomycin(FreeBase)waspurchasedfromMKbio(Shanghai,China).RecombinantmouseGM-CSF(granulocyte-macrophagecolonystimulatingfactor),IL-4(interleukin4)andIL-2(interleukin2)wereobtainedfromPeprotech(RockyHill,NJ,USA).
MP6013-500UGHumanDiI-Ac-LDL
Afterwashing,cellswereplatedonculturedishesatthedensityof5×106andculturedwithMicrovascularEndothelialCellGrowthMedium-2(EGM-2MV)BulletKit(Lonza,Walkersville,MD,USA)inahumidifiedatmosphereof5%CO2at37°C.Twodayslater,non-adherentcellswereremovedbywashingwithPBStwiceandtheculturemediumwasreplacedevery3d.Afterculturingfor1d,7d,14dand28d,EPCswereidentifiedbyopticalmicroscopeobservation,immunofluorescenceforbothDil-AcetylatedLowDensityLipoprotein(Dil-Ac-LDL;MaoKangBiotechnology,Shanghai,China)andFITClabeledUlexEuropaeusAgglutinin1(FITC-UEA-1;MaoKangBiotechnology,Shanghai,China),andflowcytometry(FACSCaliburFlowCytometry,BD,Triangle,NC,USA)forPE-Cy7conjugatedanti-mouseCD34monoclonalantibody.
AlkalinePhosphataseColorimetricAssayKitusingp-nitrophenylphosphate(pNPP)asaphosphatasesubstratewaspurchasedfromMKbioCo.Ltd.(Shanghai,China).
Thecellsweretheninfectedwiththesamevirustiteronthefollowingdaywith.8μg/mlPolybrene(MaokangCo.,Shanghai,China).At~72h.