Mechanisms of fluoroquinolone side effects 1 Non-antibiotic effects of fluoroquinolones in mammalian cells Sujan Badal1, Yeng F. Her1, and L. James Maher, III* Department of Biochemistry and Molecular Biology, Mayo Clinic 200 First Street Southwest, Rochester, Minnesota 55905 Running title: Mechanisms of fluoroquinolone side effects 1Co-first authors *To whom correspondence should be addressed: Jim Maher, Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, United States of America. Tel-507-284-9041; Fax-507-284-2053; E-mail: Maher@mayo.edu Keywords: dioxygenase; iron; antibiotic action; enzyme inhibition, epigenetics, hypoxia-inducible factor (HIF), tendinopathy Background:Fe(II)-dependentdioxygenases regulateepigeneticcontrol,collagenmaturation, and HIF degradation. Results:Ironchelationbyfluoroquinolone antibioticsresultsinDNAandhistone hypermethylation,suppressionofcollagen prolylhydroxylation, and inhibition of HIF mRNA translation. Conclusion:Dioxygenaseinhibitionmayexplain renaltoxicityandtendinopathysideeffectsof fluoroquinolones.Significance: This study suggests mechanisms for obscurefluoroquinolone-associatedsideeffects, and possible novel applications of these antibiotics as HIF antagonists. Abstract Fluoroquinolones(FQ)arepowerfulbroad-spectrumantibioticswhosesideeffectsinclude renaldamageand,strangely,tendinopathies.The pathologicalmechanismsunderlyingthese toxicitiesarepoorlyunderstood.Hereweshow that the FQ drugs Norfloxacin, Ciprofloxacin, and Enrofloxacinarepowerfulironchelators comparabletoDeferoxamine,aclinically-useful iron chelating agent.We show that iron chelation byFQleadstoepigeneticeffectsthrough inhibitionof!-ketoglutarate-dependent dioxygenasesthatrequireironasaco-factor.ThreedioxygenaseswereexaminedinHEK293 cellstreatedwithFQ.Atsub-mMconcentrations these antibiotics inhibited Jumonji domain histone demethylases,TETDNAdemethylases,and collagenprolyl4-hydroxylases,leadingto accumulationofmethylatedhistonesandDNA, andinhibitionofprolinehydroxylationin collagen,respectively.Theseeffectsmayexplain FQ-inducednephrotoxicityandtendinopathy.By the same reasoning, dioxygenase inhibition by FQ was predicted to stabilize transcription factor HIF-1!byinhibitionofoxygen-dependentHIF prolylhydroxylation.Indramaticcontrasttothis prediction,HIF-1!proteinwaseliminatedbyFQ treatment.Weexploredpossiblemechanismsfor thisunexpectedeffectandshowthatFQinhibit HIF-1!mRNAtranslation.Thus,FQantibiotics induceglobalepigeneticchanges,inhibitcollagen maturation,andblockHIF-1!accumulation.We suggestthatthesemechanismsexplaintheclassic renaltoxicitiesandpeculiartendinopathies associated with FQ antibiotics. FoodandDrugAdministration(FDA)approved antimicrobialdrugsaredesignedtotarget pathogenicmicroorganismswithminimaleffects onthehost.However,non-antibioticeffectsof antimicrobialagentsarewell-known(1),dueto unexpectedinteractionswithcellularpathways. Generalizedadverseeffects(2-4)arecommonto most antimicrobials, balancing against benefits (5-11). Here we investigate the interaction of relevant concentrations of fluoroquinolone (FQ) antibiotics Ciprofloxacin(CIPRO;Fig.1A),Norfloxacin (NOR), and Enrofloxacin (ENRO) with a cultured humanembryonickidneycellline,revealing previouslyunreportedenzymeinhibitioneffects http://www.jbc.org/cgi/doi/10.1074/jbc.M115.671222 The latest version is at JBC Papers in Press. Published on July 23, 2015 as Manuscript M115.671222Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on August 4, 2015http://www.jbc.org/Downloaded from Mechanisms of fluoroquinolone side effects 2 thatmayexplaintoxicitiesassociatedwithFQ treatment.FQarepopularsyntheticbroad-spectrum antibioticsthatexerttheirantimicrobialeffectby preventingenergy-dependentnegative supercoilingofbacterialDNAthroughgyrase inhibition(12).FQareeffectiveagentsthattarget both gram-negative and gram-positive bacteria and arerecommendedforseverebacterialinfections, includingmultidrugresistantinfections(13).FQ sideeffectshavebeenwidelystudied(14-19). However,themolecularmechanismsunderlying these toxicities remain to be elucidated.One such peculiarFQsideeffectistendinopathy(15,20). Themajority(>85%)ofFQ-associated tendinopathiesoccurwithinamonthofinitialFQ therapy,withthree-foldhigherchanceoftendon rupturewithinthefirst90daysofexposure(21). Inrarecasesofpatientswithpre-existing musculoskeletaldisorders,FQtherapyhasbeen linked to tendinopathy as early as a few hours after administrationtoaslateas6monthsafter discontinuingmedication(22).Although compromised collagen integrity after FQ treatment is well recognized in animal models (17,22,23) the underlyingmechanismisunknown.Somestudies reportassociationofenhancedmatrix metalloprotease(MMP)(23,24)orcollagenase (25)expressionassociatedwithFQ-induced tendinopathy.However,adirectlinktodefectsin collagen,aproteinthataccountsforgreaterthan 6% of muscle mass (26), is still obscure.FQ-associatednephrotoxicityisalsowell-documented(27-35).Pastclinicalstudieson patientsreceivingFQtherapyhaverevealeda strongassociationwithacuterenalfailure involvinginterstitialnephritis(27,32,34),acute tubularnecrosis(29),andmorerecently, crystalluria (33,35). These complications are often attributedtoimmune-mediatedallergic hypersensitivitytoFQantibiotics,withreversal afterdiscontinuationofdrugtreatment(31,35). AlthoughconsiderableclinicalevidenceforFQ-associatednephropathyisavailable,detailed cellulareffectsoftheseantibioticsleadingto nephritis are not well understood. Appreciating the mechanismofpathologicalsideeffectsis important for improving our understanding of FQ-associatednephrotoxicity,andforilluminating potentialcomplications.Here,weprovide evidencefornewmechanismsofFQtoxicity involving renal cell epigenetics, impaired collagen maturation,andsuppressionofhypoxia-inducible factor,HIF-1!.Weshowthatatleastsomeof these effects are due to the powerful iron-chelating property of FQ drugs. AnintrinsicFQcharacteristicisthe propensity to bind to metal cations (36-38). This is duetotheelectronegativeoxygenatomsinthe adjacentpyridoneandcarboxylatemoieties(Fig. 1) of all quinolone derivatives (39).The potential formetalchelationbyFQsuggestsmultipletoxic effects on cells. Here we focus on FQ effects on a classofFe(II)-dependentenzymesknownas2-ketoglutarate(2-KG)-dependentdioxygenases (40).Thefirstandbest-characterized2-KG dioxygenaseisprolyl-4hydroxylase,which catalyzesthepost-translationalhydroxylationof prolineresiduesincollagen(41,42).OtherFe(II)-dependentdioxygenasesincludeHIF-1!-prolyl hydroxylasedioxygenase(PHD),Jumonjidomain histonedemethylases(JMHD),andTET methylcytosine dioxygenase 1 (TET1), responsible forhydroxylationoftheHIF-1!transcription factor,histonedemethylation,andDNA demethylation,respectively.Herewetestthe hypothesisthatallofthesedioxygenasesare subjecttoinhibitionbytheironchelating properties of FQ antibiotics.Incontrasttothesedramaticepigenetic changesconsistentwiththepredictedeffectsof ironchelationondioxygenases,wereportan unpredictedresultinthecaseofHIF-1!.Here dioxygenaseinhibition shouldstabilizeHIF-1!by protectingitfromprolylhydroxylation(43).In fact, FQ treatment has the opposite effect, strongly suppressing HIF-1! accumulation. Thus,wesuggestthatironchelationby FQantibioticsinhibits!-KG-dependentcollagen prolyl-4-hydroxylaseandotherdioxygenase enzymes,perhapsexplainingFQsideeffects, includingspontaneoustendonruptures(44).In addition,FQ-inducedepigeneticmodifications uncoveredheremayexplainaspectsofFQ nephrotoxicity.Finally,ourunexpected observationofFQ-inducedHIF-1!losssuggests thepossibleuseofFQdrugsincancertherapy (45-48). EXPERIMENTAL PROCEDURES Cellculture-Humanembryonickidney (HEK293)cellswereculturedunder by guest on August 4, 2015http://www.jbc.org/Downloaded from Mechanisms of fluoroquinolone side effects 3 physiologicallyrelevantoxygenconditions(49): (37C,90%humidity,5%CO2,2%oxygen balancedbyN2)inDMEMmedium(Gibco) containing10%FBSand1% penicillin/streptomycin. Iron competition assay - The universal siderophore assayofSchwynandNeilands(50)wasusedto measuretheiron-chelatingactivityofFQ antibiotics.Deferoxaminemesylate(DFO; Calbiochem),asiderophoreproducedby Streptomycespilosus,wasusedasthepositive control.ChromeAzurolS(CAS)assaysolution (100mL)waspreparedwiththefollowingfinal concentrations:CAS(Sigma,199532;0.15mM), hexadecyltrimethylammoniumbromide(Sigma, 1102974; 0.6 mM), iron (III) chloride hexahydrate (Sigma, 236489; 0.015 mM from a stock dissolved in10mMHCl),and4.3ganhydrouspiperazine (Sigma,P45907)dissolvedin6.25mLof12M HClandadjustedthepHto5.6.Thesolutionwas storedinthedarkat4C.Ironbindingreactions wereconductedintriplicatewith0.5mLaliquots oftheCASassaysolutionandvarious concentrationsofantibioticstoatotalvolumeof 700L.Testsampleswereincubatedatroom temperaturewithslowrotationfor30min,and thentransferredtoa96-wellplateforabsorbance measurement(630nm)usingamicroplate spectrophotometer.Theapparenthalfmaximal inhibitoryconcentration(IC50)forironcomplexes wasestimatedasdescribedinthesupplemental materials. Stoichiometrydeterminationwasconductedbased on Schwyn and Neilands assay as described above. Inatypicalassay,theconcentrationofiron(Fe+3) was held constant and increasing concentrations of test compounds required to quench the absorbance ofCAS-Fecomplexwasadded.Becausetested drugconcentrationsareallwellabovethe equilibriumdissociationconstantforcomplex formation,the[compound]/[Fe+3]ratiorequired for complete quenching in such titrations gives the compound:Fe+3 binding stoichiometry. CellculturetreatmentwithFQ,DFO,CoCl2,and ferriccitrate-10mMstocksolutionsofCIPRO, ENRO,andNOR(Sigma,17850,17849,N9890, respectively)werepreparedbydissolvingFQin 0.01NHCl.HEK293cellswereculturedin10-cmdishesto70%confluencepriortotreatment withFQattheindicatedfinalconcentrationsfor theindicatedtimes.Insomeexperimentscells weretreatedwitheither100"MDFO(Sigma, D9533) or CoCl2 for 4 h. An equal volume of 0.01 NHCl(NT)wasaddedtoaseparatedishofcells asnegativecontrol.Inco-treatmentexperiments, cellswerefirsttreatedwithCIPROfor30min followedbyadditionoftheindicated concentrationsofferriccitrate(Sigma,F3388)for 4h.Cellsviabilitywasdeterminedusingtrypan blue dye exclusion. Immunoblotting-Westernblotanalyseswere performedbygrowingcellsin10-cmdishes, harvesting,andlysiswithRIPAbuffercontaining 1xproteaseinhibitorcocktail(Roche)and1x phosphataseinhibitor(ThermoScientific).Cell lysatewasagitatedonicefor20minpriorto centrifugation at 14,000 rpm for 15 min at 4 C in amicrocentrifuge.Extractswereanalyzedby electrophoresisthrough10%BIS-trispoly- acrylamindegelsunderreducingconditionswith detectionbyWesternblottingusinganti-HIF-1! antibody(BD610958,1:1000),anti-HIF-2! antibody(NB100-122,1:1000),anti-H3K9me2 antibody(Abcam1220,1:1000),anti-H3K9me3 antibody(Millipore07-442,1:500),anti-H3K27me2antibody(Abcam24684,1:1000), anti-H3K27me3antibody(Millipore05-1951, 1:15,000),anti-H3antibody(SC10809,1:1000), anti-HDAC6antibody(CS7558S,1:1000),anti-JMJD2Dantibody(Abcam93694,1:200),anti-TET1antibody(Abcam156993,1:1000),oranti-actinantibody(SigmaA2066,1:500).Secondary antibodieswereanti-rabbitandanti-mouse conjugatedtoahorseradishperoxidase(Promega, 1:15000)andsignalsweredevelopedusingan ECL plus kit (Pierce). GenomicDNAextractionandhydrolysis-A QiagengenomicDNAextractionkitwasusedto harvestgenomicDNAfromcells.The manufacturersinstructionswerefollowedwith minor changes as described (51).Briefly, the cell pelletwaslysedwithC1bufferandsubjectedto centrifugation at 1,000 rpm for 10 min at 4 C in a clinicalcentrifuge.Pelletednucleiwerere-suspendedinC1bufferwithcentrifugationfor5 min at 4C. G2 buffer was used to lyse the nuclear membranes.RNaseAsolution(Thermo Scientific,finalconcentration10mg/mL)and by guest on August 4, 2015http://www.jbc.org/Downloaded from Mechanisms of fluoroquinolone side effects 4 ProteinaseKsolution(Sigma,finalconcentration 10mg/mL)wereaddedtothelysednucleiand incubatedovernightat55C.Subsequent purificationstepswereaccordingtothe manufacturersinstructions.GenomicDNAwas washedwith70%ethanol,re-suspendedinwater, and stored at -20 C.Three microgram of genomic DNAwashydrolyzedtomononucleosidesas described(52).Theresulting40Lmixture contained3gDNA,1xmicrococcalnuclease buffer (New England BioLabs), 400 mM MgCl2, 4 mMZnCl2,20UdeoxyribonucleaseI(New England BioLabs), 2000 U micrococcal nuclease I (NewEnglandBioLabs),5Uantarctic phosphatase(NewEnglandBioLabs),and0.4U snakevenomphosphodiesterase.Reactionswere incubated overnight at 37 C. LC-MSanalysisofnucleosides-LC-MSwas performedbyloading0.6gmononucleosides from digested genomic DNA onto a C18 analytical reversephasecolumn(Phenomenex-C181.0x250 mm)usinganAgilentseries1100instrument (Agilent Technologies) with mobile phase A (0.05 M ammonium formate, pH 5.4; Sigma, 17843) and mobilephaseB(methanol)ataflowrateof0.05 mL/min and absorbance at 277 nm. The following gradient program was used: 0 min: 2% B; 18 min: 10%B;30min:25%B;35min:2%B,and60 min: 2% B.Mass spectrometry was performed as previouslydescribed(52).Briefly,theHPLC effluentwasconnectedin-linetoamass spectrometer(MSD-TOF,AgilentTechnologies) operated in positive ion mode.The MS conditions were:nebulizer20psi,dyinggas7L/min,gas temperature325C;fragmentor45V,Oct1DC 37.5V;OctRF250V.Alldatawereanalyzed usingAgilentMassHunterQuantitativeAnalysis Software. JNK1activityassay-AJNKactivityscreeningkit (Abcam,ab65784)wasusedtodetermineJNK1 enzymeactivityinanin-vitrokinasereaction. Briefly,JNKwascapturedfromtotalcelllysate usinganN-terminalc-Jun(1-79)fusionprotein boundtoglutathioneSepharosebeads.After removalofnon-specificallyboundproteinsby severalwashes,theJNK1/c-Junbeadswere incubatedinthekinaseassaybufferfor30minat 30 C in the presence of various concentrations of CIPRO.Thekinasereactionwastheninitiatedby additionofATP.C-Junphosphorylationwas measuredbywesternblotanalysisusingananti-phospho-c-Jun antibody. HSP90immunoprecipitation-Totallysate(0.4 mg)fromcontrolanddrug-treatedcellswas incubatedovernightwith5Lanti-HSP90 antibody(Abcam,13495)at4o C.ProteinA/G magnetic beads (20 L) were added to the mixture withincubation(gentlerotation)for4hat4o C. Immunoprecipitatedsampleswerewashedand subjectedtoWesternblotanalysiswithanti-pan-acetyllysineantibody(CellSignaling,94415)and anti-HSP90 antibody. Quantitativereal-timePCR-HIF-1!,HSP90, HDAC6,P4HA1,andLH1mRNAlevelswere quantifiedbyqRT-PCRanalysis.HEK293cells treated with CIPRO or DFO in 2% oxygen for 24-48hwere harvestedandtotalRNAwasextracted usinganRNeasyMicrokit(Qiagen,74004). cDNAwassynthesizedfromtotalRNAwith oligo-dT primers using SuperScript III First Strand SynthesisSystem(Invitrogen,18080-051).Primer efficiencywasdeterminedbyvalidating performancewithastandardcurve(Ctvaluevs. logDNAdilution)withacorrelationcoefficient (R2)of1correspondingto100%primer efficiency.ForsamplemRNAquantification,20 ngcDNAtemplatewaspreparedwith1M forwardandreverseprimers,andamastermix from the LightCycler TaqMan Master Kit (Roche, 04535286001)."-Actinwasusedasaninternal controlandresultswereanalyzedrelativeto#Ct values.Allcomputationsweredoneusingthe PfafflmethodforrelativeRT-PCRanalysis(53). Primer sequences were forward 5#-CAGAGCAGG AAAAGGAGTCA and reverse 5#- AGTAGCTGC ATGATCGTCTGforHIF-1!,forward5#-GCTA CTGCCATCCAATCGAGandreverse5#-CTCT CCTATGTGCTGGCCTTforVEGF,forward5#- GGTGTCTGATGATGAAGA CGAG and reverse 5#-CACCTCCAGCTCCTTCAGTTforHSP90, forward5#-CCCAATCTAGCGGAGGTAAAand reverse5#-CCTCACCTGTCATCCCAGAGfor HDAC6, forward 5#- GTGGA TTACCTGCCAGA GAGACA and reverse 5# - CTCGGCTCAGCCTT GGTTTforP4HA1,forward5#-GGAACCTG GCCTATGACACCCTandreverse5#-TGCCAT GCTGTGCCAGGAACTforLH1,forward5#- by guest on August 4, 2015http://www.jbc.org/Downloaded from Mechanisms of fluoroquinolone side effects 5 CAAATATGTACGGGGCAACC and reverse 5# - TACTCAGACCTGGGGGTACGforJMJD2D, andforward5#-GCTCTCATGGGTGTCCAATT GCT and reverse 5# - ATGAGCACCACCATCAC AGCAG for TET1. Metabolic labeling - 2 x 106 cells were seeded into 10-cmdishesandculturedfor24hat2%O2, washedandthenincubatedwithmethionine-free DMEM(Gibco)containing10%dialyzedserum for18h.Cellswerethenincubatedwiththe indicated concentrations of DFO or CIPRO for 30 min.Newly-synthesizedproteinswere radiolabeledbyadditionof5"Ci[35S]-labeled Methionine(L-[35S]-Methionine;PerkinElmer, 0.3mCi/mL).Afterthecellswerepulsedfor40 min, cells were washed twice with PBS before the addition of DMEM containing L-methionine. Cells were harvested in cell lysis buffer [50mM HEPES-KOH(pH8.0),2mMEDTA(pH8.0),150mM NaCl, 1% Triton-X, 0.1% NaDeoxycholate, 0.2 % SDS,1mMPMSFandcocktailproteaseinhibitor tablet] on ice. Total lysate (500 g) was incubated with5Lanti-HIF1!monoclonalantibody(BD Bioscience)for4hat4o C.ProteinA/Gmagnetic beads(20L)wereaddedwithincubationand gentle rotation for 4 h at 4o C. Immunoprecipitated sampleswereresuspendedinsamplebufferand heatedat95o Cfor5min.RadiolabeledHIF-1! proteinwasassessedusingSDS-PAGEanalysis and storage phosphor imaging (Typhoon system). Collagenhydroxyprolinequantification-Total hydroxylatedprolineresiduesincollagenwere determinedusingtheHydroxylprolineAssayKit (Chrondrex,6017)asdirectedbythe manufacturer.Briefly,HEK293cellswere stimulatedwith50"g/mLascorbatetostimulate collagenproduction(54,55)andtreatedat0,24, and48hwith100,150,and250MofFQor DFO.Cells were washed three times with PBS to removeanyresidualdrugpriortoeachtreatment. At72h,HEK293cellswereharvested,counted, andlysedwith100"L5NHClinpolystyrene tubes(Falcon,352058).Thecelllysatewas incubatedin60Cfor24hpriortocentrifugation at 10,000 rpm for 3 min in a microcentrifuge.The supernatant(lysate)wasanalyzedtocalculate hydroxyprolinelevelsbycolorimetricanalysis using a standard curve. RESULTS FQarepotentironchelators-Metalbindingby FQhasbeendescribed(38).Physiochemicaland spectroscopicanalysissuggesta3:1(Cipro:Fe+3) coordinationcomplexinvolvingpyridoneand carboxylateoxygenatoms(Fig.1)presentinall quinolones(39).Weverifiedandquantitatediron bindingbythreeFQantibiotics(CIPRO,NOR, andENRO).Thecolorimetricsiderophore detectionassayofSchwynandNeilands(50)was employed. FQ competition with Chrome Azurol S (CAS)forironbindingreducestheabsorbanceof theassaysolutionat630nm.Strongiron chelationwasobservedforthethreetestedFQ drugs as well as the DFO positive control (Fig. 2).ToconfirmthestoichiometryoftheFQ-iron complex,weutilizedtheSchwynandNeilands assaytodeterminetheconcentrationofFQ necessarytoquenchtheabsorbanceofaknown concentrationofCAS-Fecomplex.Analysiswas performedforCIPROandDFO(positivecontrol knowntoforma1:1complexwithiron).The results(Fig.2B-C)confirmthatDFOandCIPRO form1:1and3:1complexes,respectively(Fig. 1B-C).IC50valueswerethenestimated(Table1). ThethreetestedFQdrugschelateironatleastas well as DFO, a well-known siderophore in clinical use.Thisverificationofstrongironchelationby FQantibioticsledustoexplorethebiological effectsofFQoniron-dependentenzymesin cultured cells. FQinhibitJumonjidomainhistonedemethylase andTETdemethylaseactivitiesinculturedcells-All2-KG-dependentdioxygenasesinitially hydroxylatesubstratesusingchemistrythat requires Fe2+, 2-KG, and O2 (56,57) in ordered tri-trireactionsproducingsuccinateandCO2asby-products(40,41).DecreasesinFe2+ (58),2-KG (59),orO2(60,61)inhibittheactivitiesofthese enzymes.To assess whether iron chelation by FQ inhibits2-KG-dependentdioxygenasesincultured kidneycells(amodelofkidneyexposuretoFQ afterantibiotictreatment),wetreatedHEK293 cells with CIPRO, NOR, or ENRO for 2, 4, or 6 h inhypoxia(2%O2).HighFQconcentrations(>1 mM)weretoxicafter8h.Incontrast,cells tolerated FQ treatment for 4 h. This time point was chosenbecauseHPLCanalysisshowedmaximal druguptakeintocells(Fig.3)withoutcell by guest on August 4, 2015http://www.jbc.org/Downloaded from Mechanisms of fluoroquinolone side effects 6 detachment,andwithgreaterthan95percentcell viability as detected by trypan blue dye exclusion. WeevaluatedhistoneandDNA demethylation by the JMHD and TET1 families of dioxygenases,respectively.Thesedioxygenases playrolesindeterminingtheepigeneticstatusof chromatin.JMHDcatalyzestheremovalof methylgroupsfromhistonetails.Itsinhibition leadstoincreasedglobalhistonemethylation. TET1catalyzesthefirststepofcytosine demethylation.InhibitionofTET1resultsin global accumulation of 5-methylcytosine (5mC) in genomicDNA.Westernblotanalysisshowed accumulationofH3K9me2,H3K9me3, H3K27me2,andH3K27me3incellstreatedwith (0.11mM)CIPRO,NOR,andENROcompared to control (Fig. 4). Further, analysis of protein and mRNAexpressionforhistonedemethylase (JMD2D)andTET1showedlittleornoeffectof FQ treatment (Fig 5), confirming that loss of these activitiesisduetoenzymeinhibition.LC-MS analysis also showed an increase in 5mC levels in thegenomicDNAofcellstreatedwith0.5mM CIPRO,NOR,orENROfor4hinhypoxia comparedtocontrols(Fig.6).Importantly,these inhibitoryeffectswerereversiblebyco-treatment ofcellswithferriccitrate,pointingspecificallyto ironchelationasthemechanismofdioxygenase inhibition (Fig. 4B, 6). FQ inhibit COL-P4H enzyme activity and suppress COL-P4HandLHmRNAlevels-Collagen maturationinvolvesextensiveprolyl hydroxylationcatalyzedbyiron-dependent dioxygenases(62).Thesecollagenprolyl4-hydroxylasesarelocatedwithinthelumenofthe endoplasmicreticulumandcatalyzetheformation of4-hydroxyprolinebythehydroxylationof prolinesin-X-Pro-Gly-sequencesincollagens andmorethan15otherproteinsthathave collagen-likedomains(62-64).Thus,prolyl4-hydroxylaseshaveacentralroleinthe maturation ofcollagens,as4-hydroxyprolineresiduesare essentialfortheformationofthecollagentriple helix.ToassessFQeffectsoncollagen maturation,HEK293cellsweretreatedfor72h withincreasingFQconcentration.FQ-treated cellsshoweddecreasedcollagenprolyl- hydroxylationrelativetountreatedcells.Similarly,treatmentwithDFOshowscomparable reductionofhydroxylatedprolineresidueas expected.Additionally,co-treatmentofcellswith ferriccitrate(300"M)causedpartialreversalof effectsseenduringFQtreatment,reinforcingthe roleofironchelationindioxygenaseinhibition (Fig 7). Surprisingly,FQtreatmentalsoledto decreasedCOL-P4H1andLH1mRNAlevels (Fig.8).SimilartoP4H,lysylhydroxylases supportcollagenmaturationbyhydroxylating lysineresiduesthatserveasattachmentsitesfor galactose and glucosylgalactose, and as precursors ofthecrosslinkingprocessthatgivescollagenits tensilestrength(65).Asdiscussedbelow,FQ downregulationofCOL-P4H1andLH1mRNA levelsmayreflecttheroleofHIF-1!indriving expression of these genes (66).Theseresultssuggest,forthefirsttime, thatFQtreatmentcancauseunanticipated epigeneticeffects.Moreover,wesuggestthatthe well-establishedlinkagebetweenFQtreatment andtendinopathyreflectsimpairmentofcollagen maturationbyFQ.Wesuggestthatitisthe inhibition of collagen 4 prolylhydroxylases by FQ-mediatedironchelation,andrepressionof collagen P4H1 and LH1transcription that underlies thepeculiartendinopathysideeffectsofFQ antibiotics. UnexpectedsuppressionofHIF-1!afterFQ treatment - PHD is a 2-KG dependent dioxygenase thatdeterminesthefateofhypoxia-inducible transcriptionfactors(HIFs)incells.Innormoxia, PHDhydroxylatesHIF-1!andHIF-2!,triggering theirinteractionswiththevonHippelLindau (VHL)E3ubiquitinligasecomplexmarkingthe proteinsforproteasomaldegradation.Incontrast, stabilizationofHIF-1!andHIF-2!inhypoxia leadstoexpressionofgenesinvolvedin compensatingphysiologicalpathwayssuchas angiogenesis,glucoseutilization,cell proliferation,andtumorprogression(67).We hypothesizedthatFQinhibitionofPHDbyiron chelationwouldthereforestabilizeHIF-1!and HIF-2!.Controlexperimentsinvolvedcells treatedwithknownironantagonistsDFO(a siderophore) or Cobalt chloride (CoCl2), a divalent metalioncompetitorfordioxygenasebinding.In both cases, HIF-1! and HIF-2! levels increased as expected(Fig.9).Itwasthereforecompletely unexpected that cells treated with CIPRO, ENRO, orNORshowedaprofounddecreaseinHIF-1! by guest on August 4, 2015http://www.jbc.org/Downloaded from Mechanisms of fluoroquinolone side effects 7 and HIF-2! levels relative to controls (Fig. 9A,B).Remarkably,HIF-1!andHIF-2!weresuppressed inCIPRO-treatedcellsevenuponco-treatment with DFO or CoCl2 in hypoxia (Fig. 9C-E).These resultssuggestthatFQexertsuppressiveeffects onHIF-1!andHIF-2!proteinlevelsupstreamof regulatedproteolysis.Weconsideredthree possibleexplanationsforHIF-1!andHIF-2! suppressionbyFQ:increased proteasomal/lysosomalHIFdegradationbya VHL-E3ligase-independentmechanism, inhibitionofHIFgenetranscription,orinhibition of HIF mRNA translation. FQinhibitJNK1activity-Ithasbeensuggested that c-Jun N-terminal kinases (JNK) play a role in VHL-independentdegradationofHIFproteins (68,69).Gongetal.(68),foundthatsome quinolone-deriveddrugsboundstronglytothe ATPbindingpocketofJNK1.Zhangetal.(69) suggested a PHD-VHL-independent mechanism of HIF-1!degradationinvolvingchaperoneproteins HSP90andHSP70.Theproposedmechanismof HIF-1!lossinvolvedcompromisedJNK1 functionleadingtodestabilizationofhistone deacetylase6(HDAC6)andsubsequenthyper- acetylationofHSP90,inhibitingthechaperone (69).AsHIF-1!isaknownclientproteinof HSP90,JNK1inhibitioncouldleadtoHIF-1! misfoldingandproteasomaldegradation.To investigatethismodel,weinhibitedJNK1in HEK293cellsusingknowninhibitorSP600125 andassessedHIF-1!proteinlevelsbywestern blotting (Fig. 10A). As predicted, HIF-1! was lost upon JNK1 inhibition. We then tested if FQ inhibit JNK1 function. Using a commercial JNK1 in vitro assay,wetestedtheabilityofJNK1to phosphorylateclientproteinc-Juninpresenceof 1-100"MCIPRO.JNK1inhibitionwasobserved (Fig.10B).CIPROinhibitionofJNK1tendedto supporttheZhangetal.study(69)asaplausible explanationforFQsuppressionofHIF.However, althoughweconfirmedthatHDAC6inhibition doesleadtoHIF-1!loss(Fig.10C),thislossof JNK1activitywasnotcorrelatedwith destabilizationofHDAC6(Fig.10D-E).In contrast,theparentstudy(69)showedlossof HDAC6mRNAandproteinuponJNK1 inhibition.Further,thepredicteddownregulation ofHSP90transcriptionuponJNK1inhibitionwas notdetected(Fig.10D).Thus,whilewefound CIPROtobeaJNK1inhibitorinvitro,this property did not provide a clear link to suppression of HIF levels in cell culture. FQdonotstimulateHIFdegradationin proteasomal or lysosomal pathways - As shown in Fig.9,FQtreatmentofHEK293cellssuppresses HIF-1! accumulation independent of DFO, CoCl2, orhypoxia.WeexploredwhetherFQincreased HIF-1! degradation. We treated cells with CIPRO inthepresenceofthepotentproteasomeinhibitor MG132 (10 M). The results (Fig. 11A) show that proteasomeinhibitiondoesnotrescueHIF-1!. Thisfindingisconsistentwithourfindingsfor JNK1inhibition.Similarly,lysosomalprotease inhibitionbyleupeptin(100M)hadnoeffect (Fig.11B).Thus,HIF-1!suppressionuponFQ treatmentpresumablyinvolvesanearlierstepin HIF-1! biosynthesis. FQinhibitHIFmRNAtranslation-Todetermine if HIF-1! gene transcription is inhibited by FQ we quantifiedHIF-1!mRNAfromCIPRO-treated cellsbyqRT-PCR.NosignificantchangeinHIF-1!transcriptlevelswasobserved(Fig.11C). Combined with the prior observations that HIF-1! proteinlossdidnotresultfromincreasedprotein degradation,thisresultplacedfocuson suppressionofHIF-1!mRNAtranslationinthe presenceofFQ.EvidenceforsuchFQ-induced translationrepressionwasobservedinan experiment to measure new HIF-1! synthesis after metaboliclabeling(Fig.12).Hereweobserved thatFQtreatmentblockednewHIF-1!mRNA translationuponadditionofmethionine.Actin mRNAwastranslatedequallyinboththeFQand control cells (Fig. 12). FQ-inducedHIFmRNAtranslationrepressionis notlinkedtomicrotubuledisruptionCarbonaro etal.reportedHIF-1!translationalrepression uponmicrotubuledisruptionbycertaindrugs.Cellstreatedwithtaxol,amicrotubulestabilizer, showedlossofHIF-1!synthesisduetoHIF-1! mRNA targeting to P-bodies (70). Our analysis of microtubulestructuredidnotshowclearsignsof disruption or stabilization (Fig 13A). Furthermore, analysisofHIF-1!mRNAdistributionby immunofluorescencestudiesdidnotrevealclear by guest on August 4, 2015http://www.jbc.org/Downloaded from Mechanisms of fluoroquinolone side effects 8 aggregationpatterns(Fig13B),arguingagainst HIF-1! sequestration into P-bodies. DISCUSSION Tendinopathiesrepresentdistinctiveandpeculiar sideeffectsofFQantibiotics(71-73).This collagenpathologyhasbeenconsidered mysterious.Somestudieshavesuggested extracellularmatrixirregularitiesresultfromFQ enhancementofproteaseactivities(23-25,74,75).Here,wesuggestadifferentmechanism.FQare potentironchelatorscapableofinhibiting2-KG-dependentdioxygenasesbecauseofthecrucial roleofironintheactivesite.WeshowthatFQ treatmentinhibitscollagenmaturation.Prolyl4-hydroxylaseandlysylhydroxylaseareiron-dependentenzymesessentialforthepost-translationalmodificationofcollagen.Bothplay centralrolesincollagenmaturationthrough hydroxylationofprolineandlysineresiduesto mediatecollagencross-linking.Covalentcross-linksarerequiredforthetensilestrengthof collagenfibers(64).Wesuggestthatitisiron chelationbyFQthataccountsforsuppressed collagenhydroxylation,givingriseto tendinopathies.FQareabletochelatemultiple divalentandtrivalentmetals(37),butthe demonstrationthatepigeneticeffectsareatleast partiallyreversiblebyexogenousironsuggests thatironchelationisaprimarymechanismof inhibition. Moreover, transcript analysis of P4HA1 and LH1 show clear repression upon FQ treatment suggestingadditionalmechanismsinvolvedin collagen weakening. Invoking studies by Aro et al. (66),oursurprisingfindingofFQ-mediated suppressionofHIF-1!explainsthedecreasein P4HA1andLH1mRNAlevelsuponFQ treatment.Additionally,suppressionofHIF-1! canhavedrasticeffectsonvascularizationand energymetabolisminconnectivetissues, contributing to decreased blood flow in an already hypoxicandavasculartissue.Wesuggestthat thesethreeinsults-inhibitionofprolylandlysyl dioxygenases,reductionofP4HA1andLH1 mRNAlevels,andreducedtendonvascularization upon HIF-1! depletion together account for FQ-induced tendinopathies. WefurthershowthatbothJMHDand TETdioxygenasesareinhibitedbyFQtreatment inculturedcells,causinghistoneandDNA hypermethylation.Thisconclusionisfurther confirmedbytheobservationthatsupplemental ironpreventshistoneandDNAhypermethylation byFQtreatment.Thisisthefirststudytoshow globalepigeneticchangesinducedbyFQ antibiotics.TogetherwithFQeffectsoncollagen maturation,theseepigeneticchangesmay contributetonephrotoxicitiesobservedinpatients treatedbyFQ.Futurestudieswillbeneededto determinegeneexpressionchangesresultingfrom FQ treatment. Given their ability to inhibit dioxygenases, wereporttheunexpectedandcounterintuitive suppressionofHIF!proteinsbyFQ.WhileFQ inhibitionofPHDenzymesmarkingHIF-1!and HIF-2!fordestructionshouldstabilizethese proteins,bothHIF-1!andHIF-2!levelswere dramaticallydecreaseduponFQtreatment. Hypoxia,DFO,orCoCl2co-treatmentwasunable toovercomethissuppression.Weshowthat neitherenhancedproteindegradationnor decreasedmRNAlevelsaccountforHIF-1!and HIF-2!suppression.Instead,wefindthatHIF mRNAtranslationisinhibitedbyFQtreatment. Futureexperimentswillbenecessarytoexplore pathways associated with translational suppression of HIF-1! synthesis.Towhatextentarethepresentresults relevanttotherapeuticFQdoses?Weobserved that2-KG-dependentdioxygenaseswereinhibited byFQconcentrationsbetween10Mand1mM. CIPROconcentrationsaslowas10"Minhibited HIFmRNAtranslation.ACIPROconcentration of100"MstronglyinhibitedbothJMHDand TET1 dioxygenases, with effects even greater at 1 mM.Comparingtheseconcentrationsto physiologicalconcentrationsreportedduringFQ therapy,serumconcentrationsofCIPROare~16 M,butcanapproach1-3mMinthekidneysof treatedpatients(76).Uponoverdose,CIPRO concentrationsintheurineareevenhigher(29).Ourchoiceofhumanembryonickidneycellsto study FQ effects reflects the high kidney exposure uponFQtreatment.Indeed,acuterenalfailureis associatedwithhighFQconcentration(31).A correlationhasalsobeenobservedbetweenFQ-inducedtendinopathiesandchronicrenalfailure (77-79).Thus,weproposethatironchelationby FQantibioticsexplainstendinopathyand nephrotoxicityinpartthroughinhibitionofiron-dependent dioxygenase enzymes. by guest on August 4, 2015http://www.jbc.org/Downloaded from Mechanisms of fluoroquinolone side effects 9 Acknowledgments: WethankDouglasDerlethforproposingthehypothesistestedinthisproject,andRichardBram,Scott Kaufmann,andJustinPetersforhelpfuldiscussions.LindaBenson,MichaelHolmes,Karthikbabu Jeganathan,andEugeneKruegerprovidedimportanttechnicalassistance.Thisworkwassupportedby theMayoGraduateSchool,theMayoFoundation,andNIHgrants1F31CA180698(YFH), 5T32GM065841 (Mayo Clinic MSTP), R25 GM55252 (Mayo Clinic IMSD), and R25 GM075148 (Mayo Clinic PREP). Conflictofinterest: Theauthorsdeclarethattheyhavenoconflictofinterestwiththecontentsofthis article. Authorcontributions:SBandYFH:conceivedandexecutedexperiments.Wrotemanuscript.LJM: conceived and supervised experiments. Wrote manuscript. All authors analyzed results and approved the final version of the manuscript. by guest on August 4, 2015http://www.jbc.org/Downloaded from Mechanisms of fluoroquinolone side effects 10 REFERENCES 1.Sadarangani, S. P., Estes, L. L., and Steckelberg, J. M. (2015) Non-anti-infective Effects of Antimicrobials and Their Clinical Applications: A Review. Mayo Clin. Proc. 90, 109-127 2.Ferry,T.,Ponceau,B.,Simon,M.,Issartel,B.,Petiot,P.,Boibieux,A.,Biron,F., Chidiac, C., and Peyramond, D. (2005) Possibly linezolid-induced peripheral and central neurotoxicity: report of four cases. Infection 33, 151-154 3.Hobson-Webb,L.D.,Roach,E.S.,andDonofrio,P.D.(2006)Metronidazole:newly recognized cause of autonomic neuropathy. J. Child Neurol. 21, 429-431 4.Johannes, C. 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FIGURE1.Structuresofcompoundsandcomplexesunderstudy.(A)Ciprofloxacin.(B)Ternary chelate of CIPRO and Fe(III). (C) Deferoxamine chelate with Fe(III). FIGURE 2. FQ are potent iron chelators. (A) Iron chelation as determined by Chrome Azurol S (CAS) competition assay. Iron binding stoichiometries were determined for (B) DFO (positive control) and (C) CIPROwithironatindicatedconcentrations(higherthanKd).Dataarerepresentativeofn$3 independent experiments. NT, not treated (diluent only). FIGURE3.FQcellularuptake.(A)HPLCchromatogramofFQuptakeintoHJEK293cellsasa functionoftime.(B)StandardcurveforCIPROquantitation.(C).CalculatedintracellularCIPRO concentrations as a function of time. FIGURE 4. Accumulation of histone methylation in HEK293 cells treated with the indicated FQ. (A) H3K9me2,H3K27me2,H3K9me3,andH3K27me3abundancebywesternblottingafter4htreatment withtheindicatedFQ(0.5mM).(B)H3K27me2andH3K9me2abundanceafter2,4,and6htreatment with CIPRO (0.1 or 1 mM). (C) FQ inhibition of Jumonji domain histone demethylases in cell culture is rescued by inclusion of the indicated concentrations of ferric citrate (C6H5FeO7). NT, not treated (diluent only). FIGURE5.JMJD2DandTET1enzymelevelsinHEK293cellstreatedwiththeindicatedFQ.(A) TET1andJMJD2Dabundancebywesternblottingafter4htreatmentwiththeindicatedFQ(0.5mM). (B)TET1andJMJD2DmRNAexpressionlevelsincellsafter24hofindicatedFQtreatment.Dataare meanstandarddeviationreflectingn$3independentexperiments.Statisticalanalysisbypairedt-test (comparing to NT control: diluent only). Statistical significance (p % 0.05 **, p % 0.005 ***) FIGURE6.FQinhibitionoftheTETFe/O2/2-KGdependentdioxygenaseenzymeisrescuedafter ferriccitratetreatment.HEK293cellstreatedwithproposeddrugsorco-treatedwith300Mferric citrate. Data are mean standard deviation reflecting n$3 independent experiments. Statistical analysis by paired t-test (comparing to NT control: diluent only). Statistical significance (p % 0.05 **, p % 0.005 ***) FIGURE 7. FQ treatment inhibits collagen proline hydroxylation. HEK293 cells were co-treated with 50 g/mL ascorbate and either NT, DFO, or CIPRO for 72 h as follows: 0 h: 100 M, 24 h: 150 M, 48 h: 250 M. At 72 h, cells were harvested and processed for quantification of hydroxyproline (HyP) in total collagen. Similarly, 300 M ferric citrate was added to NT, DFO, or CIPRO cell cultures and HyP levels wereassessedaccordingly.Dataarenormalizedtocellnumberandrepresentatleastthreeindependent experiments.Dataarereportedasmeanstandarddeviation.Statisticalanalysiswasperformedusing paired t-test (comparing to NT). Significant difference (p % 0.005 ***). NT, not treated (diluent only). FIGURE8.FQrepressestranscriptionofgenesencodingenzymesinvolvedincollagensynthesis and maturation. Collagen prolyl-4-hydroxylase and lysyl hydroxylase mRNA expression levels in cells treatedwithCIPROat21%or2%O2after24htreatment.Dataaremeanstandarddeviation representative of n $ 3 independent experiments. Statistical significance (p % 0.05 **, p % 0.005 ***). NT, not treated (diluent only). FIGURE9.FQsuppressHIF-1!inHEK293cells.(A)RelativeHIF-1!levelsassessedbyWestern blotting in HEK293 cells with 0.5 mM FQ treatment for 4 h in hypoxia (2% oxygen). (B) HIF-1! levels in HEK293 cells treated with CIPRO in hypoxia. (C) HIF-1! status in HEK293 cells with co-treatment of CIPRO and DFO or (D) CoCl2 for 4 h in hypoxia. (E) HIF-2! levels in HEK293 cells with co-treatment of by guest on August 4, 2015http://www.jbc.org/Downloaded from Mechanisms of fluoroquinolone side effects 17 CIPROandDEForCoCl2 inhypoxiafor4h.100MofDFO(positivecontrol)orCoCl2(positive control) were used in the co-treatment experiments. NT, not treated (diluent only). FIGURE10.FQinhibitJNK1activity.(A)HIF-1!statusinHEK293cellsexposedtoJNK-inhibitor (JNK-1,SP600125).(B)CIPROinhibitsJNKactivity.(C)ComparisonofHIF-1!statusincellstreated withTrichostatin-A(TSA-HDACinhibitor)andCIPRO.(D/E)HDAC6/HSP90mRNAexpressionand proteinlevelsincellstreatedwithCIPRO.Dataaremeanstandarddeviationrepresentativeofn$3 independent experiments. FIGURE11.FQ-dependentreductionofHIF-1!doesnotinvolveproteasomalorlysosomal degradation.(A)Inhibitionofproteasomal(MG132)or(B)lysosomal(leupeptin)proteindegradation doesnotrescueHIF1!levelsincellstreatedwithCIPRO.(C)qRT-PCRanalysisofrelativeHIF-1! mRNA in cells treated with DFO or CIPRO. Data are mean standard deviation representative of n $ 3 independent experiments. FIGURE12.HIF-1!mRNAtranslationisrepressedinCIPRO-treatedcells.HIF-1!andactin immunoprecipitationaftermetaboliclabelingwith[35S]-Met.Cellculturesweretreatedwith1mM CIPROor100MDFOaftermethioninestarvation,thennascentproteinsradiolabeledand immunoprecipitated for further processing and imaging. FIGURE 13. FQ do not destabilize cell microtubules or cause HIF-1! sequestration in P-bodies. (A) Cellsstainedfortubulin(green)andDNA(Hoechstdye;blue).(B)StainingforDNA(Hoechstdye; blue), and HIF-1! (red molecular beacon hybridization performed as in ref. 44). by guest on August 4, 2015http://www.jbc.org/Downloaded from Mechanisms of fluoroquinolone side effects 18 Table 1 Iron binding by ligands ligandstoichiometry (ligands per Fe) Mean IC50 ("M)a Chrome Azurol S2- Ciprofloxacin352+20 Norfloxacin344+15 Enrofloxacin341+20 Deferoxamine1360+25 aMean IC50 values from n > 3 iron binding assays by guest on August 4, 2015http://www.jbc.org/Downloaded from FeO OO OO ONNH2ONH2NHNNCH3O[DFO (C25H48N6O8)] Fe(III) NNHNFOOOHNNHNFO OHONHNNFOOHOFe[CIPRO (C17H18FN3O3)]3 Fe(III) NHNNFO OOHCIPRO (C17H18FN3O3)AB C Fig. 1 by guest on August 4, 2015http://www.jbc.org/Downloaded from 0.00 0.10 0.20 0.30 0.40 0.50 0.010.1110 Vehicle DFO CIPRO NOR ENRO 0120100200300 0 1 2 050010001500 [compound] ("M) A B[Fe+3] = 311 "M [Fe+3] = 500 "M Fig. 2 C 101001000 10000 DFO CIPRO Absorbance 630nm ! NT 2 1 0 2 1 0 by guest on August 4, 2015http://www.jbc.org/Downloaded from 0 100 200 300 400 500 600 700 800 900 1000 024681012 mV Time (min) 4h 2h 6h 8h A 1.0E+00 4.0E+06 8.0E+06 1.2E+07 00.10.2 Area [CIPRO] (mM) B CIPRO (1mM) treatment Total Intracellular [CIPRO] (mM) 2 h0.562 4 h0.655 6 h0.426 8 h0.317 C Fig. 3 by guest on August 4, 2015http://www.jbc.org/Downloaded from DFO (0.1 mM) CIPRO (0.5 mM) NOR (0.5 mM) ENRO (0.5 mM) A C CIPRO (0.5 mM) Ferric-citrate ("M) H3K9me2 H3K27me2 H3 H3K9me3 H3K27me3 ++++-- --100300200100 Fig.4 NT H3K9me2 H3 H3K27me2 H3K9me3 H3K27me3 2 h 4 h 6 h CIPRO (1 mM) (0.1 mM) H3K9me2 H3 B 6 h NT NT H3K27me2 by guest on August 4, 2015http://www.jbc.org/Downloaded from 0.0 0.5 1.0 Relative mRNA Expression levels JMJD2D TET1 NTDFOCIPRO NOR ENRO NT DFO (0.1 mM) CIPRO (0.5 mM) NOR (0.5 mM) ENRO (0.5 mM) Actin JMJD2D TET1 ***** **** Fig.5 AB by guest on August 4, 2015http://www.jbc.org/Downloaded from Fig. 6 0 1 2 3 4 5 6 Percentage of 5-mC in genomic DNA ** NT DFO CIPRONORENRO

** [drug] (mM) 0.0 0.10.50.50.5 ** *** *** Fe+3 (300 "M) by guest on August 4, 2015http://www.jbc.org/Downloaded from Fig. 7 0 20 40 60 80 100 HyP level (ug/ml) *** *** NT DFOCIPRO Fe3+ (300 "M) by guest on August 4, 2015http://www.jbc.org/Downloaded from 0.25 0.50 1.00 2.00 4.00 8.00 Relative mRNA Expression (Log2) COL-Prolyl-4-Hydroxylase Lysyl Hydroxylase CIPRO (21% O2) CIPRO (2% O2) NTDFO 0.5 0.25 0.10NTDFO0.5 0.25 0.10 Fig. 8 *** *** ** *** *** ** *** *** by guest on August 4, 2015http://www.jbc.org/Downloaded from Actin HIF1# NT DFO (0.1 mM) CIPRO (0.5 mM) NOR (0.5 mM) ENRO (0.5 mM) A B C E Actin HIF1# [CIPRO] (mM)-10.10.01 [CIPRO] (mM) DFO HIF1# Actin - - ++++ -10.10.011- CoCl2

Actin HIF1# [CIPRO] (mM) - - ++++ -10.10.011- CIPRO CoCl2

DFOHIF2# Actin ++--- ++--- --+-+ Fig. 9 D by guest on August 4, 2015http://www.jbc.org/Downloaded from C Actin HIF1# [JNK-I] ("M) A 5012.525 -[CIPRO] ("M) [TSA] ("M) Actin HIF1# - - -1005011 0.5- - - -Actin HDAC6 HSP90 [CIPRO] (mM) E - 10.10.01 0.0 0.4 0.8 1.2 Relative mRNA Expression HSP90HDAC6 D [CIPRO] (mM) 1 0.1 0.010 0.010.110 Fig. 10 JNK1 P-c-Jun [CIPRO] ("M) 1001 10 - - + + + -+ ATP B by guest on August 4, 2015http://www.jbc.org/Downloaded from MG132 (10 "M)Actin HIF1# Actin HIF1# CIPRO (0.5mM) [Leupeptin] ("M)A B [CIPRO] (mM)10.10.011 - - ++++ - -- - ++++ 100 1005010- -Fig. 11 0.00 0.40 0.80 1.20 Relative mRNA Expression HIF-1#0DFO10.10.01[CIPRO] (mM) C by guest on August 4, 2015http://www.jbc.org/Downloaded from 75 100 -HIF-1# 120 kDa -Actin 45 kDa 50 37 25 Fig. 12 by guest on August 4, 2015http://www.jbc.org/Downloaded from "# $%&'( )*+,-./ B A Fig. 13 by guest on August 4, 2015http://www.jbc.org/Downloaded from IIISujan Badal, Yeng F. Her and L. James Maher in mammalian cellsNon-antibiotic effects of fluoroquinolonesEnzymology: published online July 23, 2015 J. Biol. Chem. 10.1074/jbc.M115.671222 Access the most updated version of this article at doi: . JBC Affinity Sites Find articles, minireviews, Reflections and Classics on similar topics on the Alerts: When a correction for this article is posted When this article is cited to choose from all of JBC's e-mail alerts Click here http://www.jbc.org/content/early/2015/07/23/jbc.M115.671222.full.html#ref-list-1This article cites 0 references, 0 of which can be accessed free at by guest on August 4, 2015http://www.jbc.org/Downloaded from