Research Article Apocynin and Diphenyleneiodonium Induce

Research ArticleApocynin and Diphenyleneiodonium InduceOxidative Stress and Modulate PI3KAkt and MAPKErkActivity in Mouse Embryonic Stem Cells

Jan KuIera1 Lucia Binoacute123 Katelina Štefkovaacute1 Josef Jaroš3 Ondlej VašiacuteIek24

Josef VeIela1 Lukaacuteš Kubala24 and Jiliacute Pacherniacutek14

1 Institute of Experimental Biology Faculty of Science Masaryk University Kotlarska 2672 61137 Brno Czech Republic2Institute of Biophysics Academy of Sciences of the Czech Republic Kralovopolska 2590135 61200 Brno Czech Republic3Department of Histology and Embryology Faculty of Medicine Masaryk University Kamenice 7535 62500 Brno Czech Republic4International Clinical Research Center Center of Biomolecular and Cellular Engineering St Annersquos University HospitalPekarska 53 65691 Brno Czech Republic

Correspondence should be addressed to Jirı Pachernık jipascimunicz

Received 12 August 2015 Accepted 13 September 2015

Academic Editor Tomris Ozben

Copyright copy 2016 Jan Kucera et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Reactive oxygen species (ROS) are important regulators of cellular functions In embryonic stem cells ROS are suggested toinfluence differentiation status RegulatedROS formation is catalyzed primarily byNADPH-dependent oxidases (NOXs) Apocyninand diphenyleneiodonium are frequently used inhibitors of NOXs however both exhibit uncharacterized effects not related toNOXs inhibition Interestingly in our model of mouse embryonic stem cells we demonstrate low expression of NOXs Thereforewe aimed to clarify potential side effects of these drugs Both apocynin and diphenyleneiodonium impaired proliferation of cellsSurprisingly we observed prooxidant activity of these drugs determined by hydroethidine Further we revealed that apocynininhibits PI3KAkt pathway with its downstream transcriptional factor Nanog Opposite to this apocynin augmented activity ofcanonical Wnt signaling On the contrary diphenyleneiodonium activated both PI3KAkt and Erk signaling pathways withoutaffecting Wnt Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signalingpathways

1 Introduction

Reactive oxygen species (ROS) playmultiple roles in the biol-ogy of the cell [1] NADPHoxidase (NOX) and oxidative reac-tions on the mitochondrial membrane are the main sourcesof ROS although it can also be produced by other enzymaticand nonenzymatic sources [2 3] NOX is a membrane-bound protein complex generating superoxide anion (O

2

∙minus)

from molecular oxygen which initiates the cascade of freeradical reactions in response to various stimuli Productionof ROS by NOX family has long been considered a uniqueproperty of phagocytic cells which utilize this enzyme as apart of host defense immune system Currently the regulatedROS production in nonphagocytic cells by NOX was linked

to regulation of different processes including proliferationmigration differentiation immunomodulation and oxygensensing and therefore its expression and activity are tissuespecific and are tightly controlled [4 5] On the basis of thehomology to the catalytic subunit of the original phagocyticNOX (gp91phox or preferablyNOX2) otherNOX isoformsmdashNOX1 NOX3 NOX4 and NOX5mdashhave been identified innonphagocytic cells In parallel two other members of theNOX family were discovered namely dual oxidases 1 and 2(DUOX 1 2) initially also referred to as thyroid oxidases [6]

Despite studies suggesting the importance of NOXs ingeneral the involvement of individual NOX family membersin specific function is still not completely understood One ofthe reasons is a lack of highly specific inhibitors that would

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 7409196 14 pageshttpdxdoiorg10115520167409196

2 Oxidative Medicine and Cellular Longevity

reliably block particular NOX The most frequently usedinhibitors employed in experiments are vanillin derivative 4-hydroxy-3-methoxyacetophenone (trivial names apocyninor acetovanillone APO) and diphenyleneiodonium chloride(DPI) Both of these drugs were applied in numerous in vitroand in vivo studies and although their effect was attributedprimarily to NOX inhibition specificity of APO and DPIremains questioned [7]

The proposed molecular mechanism of APO-mediatedNOX inhibition is not fully understood but it involves impair-ment of NOX complex assembly and activation [8] APO wasalso shown to act directly as ROS scavenger [9] Contrarily tothis finding other studies suggest that APO is rather a proox-idant stimulating ROS production [10ndash12] Further APO wasalso shown to modulate the generation of arachidonic acid-derived inflammatory mediators [13]

DPI was reported not only to affect NOX but also tointerfere with other flavoenzymes including nitric oxidesynthase and xanthine oxidase [14 15] Inhibitory effects ofDPI onmitochondrial ROS production were also shown [16]On the other hand DPI induces O

2

∙minus mediated apoptosis[17] inhibits cell redox metabolism and promotes generaloxidative stress [18] DPI is also suggested as a nonselectiveblocker of ionic channels [19] The above mentioned nonspe-cific effects are thought to be responsible for contradictoryresults obtained using these inhibitors Further studies areneeded to better understand the actions of APO and DPI notdirectly related to NOX inhibition

Intracellular formation of ROS leading to overall redoxstatus modulation is important for regulation of pluripotentcells differentiation [20] In mouse embryonic stem cells(mES) pluripotent cells derived from inner cell mass ofblastocyst redox alterations are thought to play a role in thebalance between self-renewal anddifferentiationUndifferen-tiated mES have several times lower ROS level in comparisonwith differentiating mES [21] It was shown that short termincrease of the ROS favors differentiation into cardiomy-ocytes [22 23] and into endoderm and mesoderm lineage[24]

Several signaling pathways are crucial for the regulationof self-renewal and differentiation ofmES Primarily themESpluripotency is controlled by Stat3 together with PI3KAktsignaling pathways [25ndash27] Importance of PI3K signalingwas demonstrated in many studies where its inhibitionnegatively regulates the self-renewal inmES [28 29] FurtherMAPKErk signaling pathway is rather important for mESdifferentiation as its inhibition improves maintenance ofthe pluripotent stem cell phenotype [30] A growing bodyof evidence indicates that Wnt120573-catenin signaling pathwayplays a vital role in the regulation of mES fate [31] Signalingpathways including Stat3 PI3KAkt Erk and Wnt are mod-ified by ROS production [5 32 33] and therefore might beaffected by NOX or other ROS modulating agents althoughthe importance of this phenomenon in mES remains elusive

Our study demonstrates unexpected prooxidant activityof APO and DPI in undifferentiated mES together withimpairment of cell proliferation Further we show that APOinhibits PI3K signaling accompanied by decrease in proteinlevel of critical ES pluripotency regulator Nanog whereas

DPI enhances both PI3K and Erk activity Interestingly APOstrengthens Wnt activity pointing out another unknownmechanism of APO-mediated changes in signaling cascadesregulating mES In contrast to PI3K and Erk we did notobserve any effect of APO or DPI on Stat3 phosphorylationwhich is considered to play the major role in mES mainte-nance

2 Material and Methods

21 Cell Culture and Treatment A feeder-free adapted mESline R1 was propagated as described previously [29] in anundifferentiated state by cell culturing on tissue cultureplastic coated by gelatin (01 porcine gelatin solution inwater) in Dulbeccorsquos modified Eaglersquos medium (DMEM)containing 15 fetal bovine serum (FBS) 100 IUmL peni-cillin 01mgmL streptomycin 1x nonessential amino acid(all from Gibco-Invitrogen UK) and 005mM 120573-mercap-toethanol (Sigma-Aldrich USA) supplemented with 5 ngmL of leukemia inhibitory factor (LIF Chemicon USA) re-ferred to here as the complete medium

APO DPI hydrogen peroxide (H2O2) N-acetylcysteine

(NAC) and LY294002 (LY) were provided from Sigma-Aldrich Stock solutions of APO (04M) DPI (10mM) andLY (10mM) were prepared by dissolving the compounds indimethyl sulfoxide Aliquots were stored at minus20∘C NAC wasprepared as a 05M stock solution in serum-free DMEMmedium pH was adjusted to 74 and filter-sterilized aliquotswere stored at minus20∘C Drugs were added directly to the incu-bation medium or freshly prediluted in sterile phosphate-buffered saline (PBS) to desired concentration

22 Cell Proliferation The cell proliferation was determinedby estimation of overall cellular protein mass in wholecell lysates that reflects the cell number as demonstratedpreviously [34] ES cells were seeded to 24-well plate incomplete media at density 5 000 cells per cm2 Next day thecells were treated with drugs for further 48 hours Finally thecells were washed twice with PBS and lysed in SDS buffer(50mM Tris-HCl pH 75 100mM NaCl 10 glycerol 1SDS 1mM EDTA) Protein concentration was determinedusing DC protein assay (Bio-Rad USA) kit according tomanufacturerrsquos instructions

23 Determination of NOX Expression Total RNA wasextracted using the UltraClean Tissue amp Cells RNA IsolationKit (MO BIO Laboratories USA) for ESC and RNAzol RT(Molecular research center Inc USA) for mouse tissuesaccording to manufacturerrsquos instructions 1 120583g of total RNAwas used for cDNA synthesis with DyNAmo cDNA Syn-thesis Kit (Finnzymes Finland) according to the manufac-turerrsquos instructions qPCR was performed in LightCycler 480instrument using LightCycler Probes Master with UniversalProbe library probes (all from Roche Germany) accord-ing to manufacturerrsquos instructions Ribosomal protein L13A(RPL13A) was used as reference gene data are presented as

Oxidative Medicine and Cellular Longevity 3

2minus(Cq(target)minusCq(reference)) The primers and probes used were asfollows

NOX1 51015840tggattttctaaactaccgtctcttc 51015840caaagtttaatgctgcat-gacc31015840 20 NOX2 51015840tgccaacttcctcagctaca31015840 51015840gtgcacagc-aaagtgattgg31015840 20 NOX3 51015840tgaacaagggaaggctcatt31015840 51015840cat-cccagtgtaaagctatgtga31015840 20 NOX4 51015840catttgaggagtcactgaact-atga31015840 51015840tgtatggtttccagtcatccag31015840 5 DUOX1 51015840acagatggg-gcagcaaag31015840 51015840gctggtagacaccatctgcat31015840 20 DUOX2 51015840cag-acagctttttgctcaggt31015840 51015840cacttgctgggatgagtcc31015840 64 RPL13A51015840catgaggtcgggtggaagta31015840 51015840gcctgtttccgtaacctcaa31015840 25

24 Analysis of ROS Production in Live Cells by AutomatedTime-Lapse Image (Live Imaging Fluorescent Microscopy)The cells were seeded to 96-well plate designed for live imag-ing fluorescence microscopy (Greiner Bio-one Germany)After 24 hours cells were pretreated with tested drugs for 15minutes and loaded with hydroethidine (HE 5120583M Sigma-Aldrich) Live imaging of prepared samples was performed at37∘C and 5CO

2atmosphere using the high-content screen-

ing microscope ImageXpressMicroXL (Molecular DevicesUSA) Seven images per well were acquired during 120minutes with scanning interval of 10 minutes By Gaussianthresholding of the HE fluorescence images mask area ofthe viewfield covered with metabolizing cells was detectedusing the Otsu method of fitting individual objects The10640 images for every measured plate were analyzed withCellProfiler [35] running on processor cluster provided byMetaCentrum (The National Grid Infrastructure) Intensityof fitted regions was lowered by minimal intensity of anappropriate image to subtract the background and the meanintensity per individual well was then calculated and visu-alized with Knime [36] using HCS tools and R StatisticsIntegration extensions [37]

25 High-Performance Liquid Chromatography (HPLC) Anal-ysis of ROS Production The HPLC detection of O

2

∙minus

was based on the detection of a specific product 2-hydroxyethidium 2-OH-E(+) which is formed in the reactionof O2

∙minus with HE [38 39] Besides specific 2-OH-E(+) also anonspecific product of hydride acceptors withHE ndash ethidium(E+) was detected The cells were seeded to 6-well plate andbefore the treatment the medium was changed for DMEM(without phenol red and sodium pyruvate) with 1 FBS Thecells were treated with APO and DPI for 120 minutes in total30 minutes before the end of the experiment HE in finalconcentration 10 120583M was added The medium after centrifu-gation was stored for optional HPLC analysis To extract theHE products ice cold methanol was added to the cells [40]for 15 minutes at 4∘C in dark shaking The supernatant wastransferred to an Eppendorf tube and centrifuged A 75120583Lsample was injected into the HPLC system (Agilent series1100) equipped with fluorescence and UV detectors (Agilentseries 1260) to separate the 2-OH-E(+) product Fluorescencewas detected at 510 nm (excitation) and 595 nm (emission)The mobile phase consisted of H

2OCH

3CN Kromasil C18

(46mmtimes 250mm) columnwas used as the stationary phaseElution conditions for the analysis of HE and its productswere used from Nature Protocols [38]

26 Western Blot Analysis Western blot analysis cell sampleharvesting and preparation were performed by a standardprocedure as presented previously [29] We used the fol-lowing primary antibodies against Nanog 120573-actin (AbcamUSA) p-Akt (S473) Akt p-Stat3 (Y705) Stat3 Erk12 p-Erk12 (T202Y204) and p-GSK3120573 (S9) (all from Cell Sig-naling Technology USA) Following immunodetection eachmembranewas stained by amido black to confirm the transferof the protein samplesThe total level of 120573-actin was detectedas loading control

27 Cell Transfection and TOPflash Luciferase Reporter AssayCells were transiently transfected using polyethyleneimine ina stoichiometry of 4 120583L per 1 120583g of DNA Super8X TOPflashconstruct Renilla luciferase construct and expression vec-tor for mutant nondegradable 120573-catenin and S33-120573-catenin(codon 33 substitution of Y for S generously provided byProfessor Korswagen) were used in concentration of 05120583gper well in a 24-well plate 24 hours after seeding 6 hoursafter transfectionmediumwas changed and cells were treatedwith NOX inhibitors or LY for 24 hours For cell stimulationWnt3a or control conditioned medium [41] was added 8hours before harvest Dual-Luciferase assay kit (PromegaUSA) was used according to the manufacturerrsquos instructionsfor the evaluation of luciferase activity Relative luciferaseunits were measured on a plate luminometer Chameleon V(Hidex Finland) and normalized to the Renilla luciferaseexpression

28 Statistical Analysis Data are expressed as mean +standard error in the mean (SEM) Statistical analysis wasassessed by 119905-test or by one-way analysis of variance ANOVAand BonferronirsquosMultiple Comparison posttestThe values of119875 lt 005 were considered statistically significant (lowast119875 lt 005lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001)

3 Results

31 Expression of NOXs in mES Is Relatively Low To confirmthe assumption of the negligible presence of NOX andDUOXhomologues in the undifferentiatedmES the gene expressionwas compared to the selected mouse tissues Particularorgans were chosen based on the described presence of NOXhomologues by various authors [4 6] In agreement with theliterature all determined NOX homologues NOX1 NOX2NOX3 NOX4 DUOX1 and DUOX2 showed two to threeorders lower expression in undifferentiated mES comparedto selected tissues (Figures 1(a)ndash1(f)) The comparison of theNOXs relative expression in mES revealed the NOX4 expres-sion to be the highest approximately 10 times compared toother NOXs (Figure 1(g)) altogether it can be concluded thatthe expression of all NOXs except NOX4 is very low

32 APO and DPI Affect mES Proliferation Both APO andDPI affected growth of mES in dose-dependent manner inthe concentration range 01 025 05 10 and 20mM forAPO (Figure 2(a)) and 10 20 40 80 160 and 320 nM forDPI (Figure 2(b)) Data showed decrease in cell proliferation


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Figure 1 Continued


NOXs in mES

NOX1 NOX2 NOX3 NOX4 DUOX1 DUOX200000

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expr

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(g)

Figure 1 Relative expression of NOX1 (a) NOX2 (b) NOX3 (c) NOX4 (d) DUOX1 (e) and DUOX2 (f) mRNA in mES and selected tissuesComparison of relative expression of individual NOXs and DUOXs mRNA within mES cells is also shown (g) Data are presented as mean +SEM from at least two independent experiments

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Figure 2 Effect of APO (a) and DPI (b) on mES proliferation after 48 h treatment based on total cellular protein mass Data represent mean+ SEM from four independent experiments Statistical significance was determined by ANOVA post hoc Bonferronirsquos Multiple Comparisontest 119875 lt 005 The groups marked differently by symbol letters are statistically significantly different from each other

using concentration from 05mM (APO) and from 20 nM(DPI) after 48-hour treatment IC50 of APO andDPI formESdetermined from these data were 11 plusmn 02mM and 185 plusmn32 nM respectively

33 APO and DPI Do Not Inhibit but Potentiate Formationof ROS in mES Despite the very low expression of NOXsenzymes in undifferentiated mES the ROS production wasdetectable in our mES lines Contrary to the expectationstreatment by APO or DPI significantly induced generationof ROS in mES cells detected by live imaging analysisof HE fluorescence continuously for up to 120 minutes(Figures 3(a) 3(b) and 3(c)) To confirm the specificityof this determination the formation of specific product ofHE reaction with O

2

∙minus the 2-OH-E(+) and also nonspe-cific product ethidium (E+) were determined by HPLC

(Figures 3(d) and 3(e)) This analysis shows potentiation ofnonspecific HE oxidation rather than O

2

∙minus formation in DPItreated cells but not in APO treated cells

34 Effect of APO andDPI on Stat3 Akt and Erk Phosphoryla-tion inmES To investigate the effect of short term treatmentmES were serum and LIF starved for 12 hours and treatedby APO and DPI for 20 minutes followed by 20-minutestimulation with FBS or LIF APO treatment resulted indecrease of Akt phosphorylation in every condition withouteffect on Erk DPI had no significant effect on Akt and Erkkinases signaling Level of p-Stat3 remained unchanged byNOX inhibitors (Figure 4)

To further clarify the dose-dependent effect of testeddrugs we employed the same experimental design withserum starvation and FBS activation Moreover glutathione


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Figure 3 ROS production inmES cells treated by 1mMAPO and 100 nMDPI computed from the automated time-lapse image acquisition ofHE fluorescence for 120 minutes (a b) selected single time point 60 minutes (c) HPLC determination of specific 2-OH-E(+) and nonspecificproduct E(+) of HE oxidation in the presence of 1mMAPO and 100 nMDPI (d e) Data are presented as mean + SEM from four independentexperiments Statistical significance was determined by ANOVA post hoc Bonferronirsquos Multiple Comparison test 119875 lt 005 The groupsmarked by an asterisk are statistically significantly different from control


Stat3

p-Stat3

p-Akt

p-Erk

Akt

Erk

LIF FBSSF

APOCtr DPI APOCtr DPI APOCtr DPI

120573-actin

Figure 4 Effect of APO (2mM) andDPI (100 nM) on the Stat3 Aktand Erk phosphorylation in serum starved mES cells (SF) treatedby drugs for 20 minutes followed by LIF (5 ngmL) or FBS (15)stimulation for 20 minutes Total level of 120573-actin was used as aloading control A typical representative western blot is shown

precursor NAC and H2O2were used as a bona fide antiox-

idant and a prooxidant respectively to distinguish betweeneffects mediated via redox changes in cultivated mES andROS independent actions of APO and DPI

APO abolished phosphorylation of Akt in a dose-dependent manner with supportive effect on phosphory-lation of Erk in higher concentrations In contrast DPIslightly increased phosphorylation of Akt and also upregu-lated phosphorylation of Erk in the highest concentrationConsistent with expectations H

2O2increased both Akt

and Erk phosphorylation NAC had no effect on signalingin this setup (Figure 5(a)) To test whether the effect onsignaling was also preserved during cultivation in completemedium mES were treated by the same concentration ofdrugs for 1 hour In this case similarly to previous treatmentAPO inhibited Akt phosphorylation in a dose-dependentmanner Erk phosphorylation was impaired in the presenceof the highest concentration of APO Notably in this setup100 120583M concentration of DPI strongly induced both Akt andErk phosphorylation Contrary to the previous setup NACdecreased phosphorylation of both pathways (Figure 5(b))

Finally we examined the impact of APO and DPI onAkt and Erk activity after 24 hours in absence or presence ofNAC (Figure 6) Contrary to the effect observed after a shortterm treatment Akt phosphorylation was upregulated whenAPO was employed Phosphorylated form of Akt was alsoincreased following the DPI treatment This activation couldbe prevented by addition of NAC Erk phosphorylation wasslightly decreased by APO treatment and augmented by DPIeven in the presence of NAC APO strongly downregulated

Nanog protein level in mES independently of NAC sup-plementation (Figure 6) Level of Stat3 phosphorylation wasmodulated in the above mentioned experimental conditionby neither APO nor DPI treatment (data not shown) We didnot observe any effect of H

2O2on the evaluated signaling

pathways after 24 hours (data not shown)

35 APO Augments Canonical Wnt Activity in mES Toassess the effects of APO and DPI supplementation on activ-ity of Wnt pathway we used TCFLEF reporter gene assay(TOPflash) to determine the level of canonicalWnt activationmediated by 120573-catenin which specifically induces the tran-scriptional activity of TCFLEF [42] Firstly we examinedour system with addition of Wnt3a conditioned media orexogenous nondegradable 120573-catenin both known agoniststo promote its activation These interventions induced tran-scription activity of reporter gene 45 and 25 times respec-tively (Figure 7(a))

PI3K inhibitor LY294002 (LY) and both NOXs inhibitorsAPO and DPI did not change the spontaneous 120573-cateninmediated transcription activity of TCFLEF (Figure 7(b)) Incontrast when the cells were treated by Wnt3a conditionedmedia presence of LY and APO significantly augmented theTCFLEF transcription activity (Figure 7(c)) On the otherhand DPI had no effect (Figure 7(c)) However all testeddrugs did not have any effect in the presence of exogenousnondegradable 120573-catenin (Figure 7(d))

To further clarify the effects of LY APO andDPI onWnt120573-catenin signaling theGSK3120573 S9 phosphorylation allowingaccumulation of 120573-catenin was evaluated [43] Treatmentwith both APO and LY but not DPI decreased GSK3120573 S9phosphorylation in this setup as shown by the western blotanalysis (Figure 7(e))

4 Discussion

APO and DPI are the most commonly used inhibitors ofNOX involved in numerous studies despite the increasingevidence questioning their specificityWe employedmES as amodel to analyze effects of these compounds that might notbe directly mediated through impairment of NOXs becauseof their generally negligible expression in undifferentiatedmESWe aimed to investigatemodulation of ROS productionby APO andDPI inmES as well as interactions with signalingpathways important for stem cell regulation

AlthoughNOXswere attracting attention as an importantsource of intracellular ROS production for a long time theirrole in stem biology remains poorly understood Previouslyit was demonstrated that NOXs expression is preciselyregulated during embryonic stem cell differentiation intocardiomyocytes [44 45] and vascular smooth muscle lineage[46]

In our experiments we observed generally low level ofNOXsDUOXs expression close to the limit of detectionwhichwe concluded both from real Cq values of PCR amplifi-cation and from comparison of NOXsDUOXs expression inseveral employed tissues As such control sample tissues withwell described NOXsDUOXs expression and activity were


p-Akt

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(a)

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120573-actin

H2O2

(b)

Figure 5 Effect of APO (1 2 4mM) DPI (01 1 100120583M) NAC (5 10 20mM) and H2O2(05 1mM) on the Akt and Erk phosphorylation

in serum starved mES cells treated by drugs for 20 minutes followed by 15 FBS stimulation for 20 minutes (a) and cells treated for 1 hour incomplete medium (b) Total level of 120573-actin was used as a loading control A typical representative western blot is shown

NAC

p-Akt

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APOCtr DPI APOCtr DPI

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Figure 6 Effect of APO (1mM) and DPI (10 nM) in absenceand presence of antioxidant NAC (10mM) on the Akt and Erkphosphorylation andNanog protein level inmES cells after 24 hoursin complete medium Total level of 120573-actin was used as a loadingcontrol A typical representative western blot is shown

used [4 6] Among NOXs and DUOXs NOX4 expressionwas the highest in mES which is in agreement with otherauthors [46 47] It may also correspond to relative abundantexpression of this enzyme across other tissues [48] Howeverit should be emphasized that level of NOX4 transcript inmESwas still nearly three orders below expression in selected

control tissue (kidney) NOX4was shown to be constitutivelyactive and hence its activity is mostly regulated by the levelof its expression [49] Concerning other potential sources ofintracellular ROS it is also noteworthy that favored relianceon anaerobic glycolysis in undifferentiated mES leads toreduced mitochondrial biogenesis and activity manifestedby declined ROS production [50 51] Thus mES can beconsidered not only NOX-low but also overall ROS-lowmodel

Next we aimed to assess effect of APO and DPI on pro-liferation of undifferentiated mES Selection of used concen-trations was based on comprehensive literature search APOas NOX inhibitor was used in range 30ndash1200 120583M [9 52] withthe upper range of these concentrations corresponding toAPO supplementation preferably employed in our experi-ments Regarding DPI many authors used concentrationsranging 1ndash100 120583M summarized in [53] which is approxi-mately one order of magnitude higher than doses used in ourexperiments as we observed significant growth impairmenteven when concentrations as low as 20 nM were appliedImpact on cell proliferation after NOX inhibitors treatmentwas described earlier for both normal and transformed celllinesThe observed effects of DPI and APOwere suggested tobe attributed to the variousmechanisms including changes inNOX mediated ROS production downregulation of integrinexpression cell cycle arrest or modulation of mitogenic-signaling pathways [54ndash57] Therefore we can assume thatdirect modulation of ROS production could contribute to theobserved decrease ofmES proliferation At the same time theeffects of inhibitors employed in this study can also be relatedto their direct effects on the other promitogen cell signalingpathways as discussed laterThe potential of APO and DPI toinhibit cell proliferation could also be beneficial in the contextof anticancer agents A recent publication showed that APOsuppressed prostate cancer and that the reduction of Rac1 andNF120581B phosphorylation was involved [58]

Further we assessed how NOX inhibitors affected ROSproduction in our model Due to their natural short half-life and high reactivity precise detection of ROS represents


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Figure 7 Effect of APOandDPI onWnt pathway activity determined byTOPflash assay andGSK3 phosphorylation on S9 Effect of theWnt3aconditioned media or exogenous nondegradable 120573-catenin on the transcriptional activation of a reporter gene (a) Effect of LY (10120583M) APO(1mM) and DPI (10 nM) on the spontaneous (b) Wnt3a conditioned media induced (c) and exogenous nondegradable 120573-catenin-inducedtranscriptional activation (d)Data representmean+ SEM fromat least four independent experiments Statistical significancewas determinedby ANOVA post hoc Bonferronirsquos Multiple Comparison test (lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) Effect of LY APO andDPI on GSK3120573(S9) phosphorylation (e) Total level of 120573-actin was used as a loading control A typical representative western blot is shown

a tremendous challenge especially in nonphagocytic cellsWe were aware of limits in ROS measurement with respectto specificity and generation of possible artefacts there-fore we used two different assays live imaging fluorescentmicroscopy and HPLC both utilizing ROS-sensitive probeHE The reaction between O

2

∙minus and HE generates a highlyspecific red fluorescent product 2-hydroxyethidium 2-OH-E(+) However in the intracellular milieu the presence ofredox metal ions or hemeproteins with peroxidase activityor other one-electron oxidants can oxidize HE to severalnonspecific products including the ethidium E(+) and

dimeric products [38 59 60] Despite the expectations thelive imaging assay showed significant prooxidant activityof APO and DPI when continuous nonoscillating probeoxidation was determined by live imaging On the otherhand HPLC analysis used for determination of specific HEderivatives did not confirm O

2

∙minus production after APO andDPI treatment The only observed effect was significant DPI-mediated elevation of E(+) APO did not induce generationof HE oxidation products both 2-OH-E(+) and E(+) whichis partially in contrast with the results from live imagingfluorescent microscopy where all nonspecific HE oxidation


fluorescent products are summarized [60] This suggests thatother oxidants rather than O

2

∙minus are responsible for increasein HE fluorescence

Although NOX inhibitors should generally relieve oxida-tive stress several lines of evidence for APO and DPIexist demonstrating the opposite It was reported that DPIinhibits pentose phosphate pathway (PPP) responsible for thesynthesis of NADPH a redox cofactor important for manyantioxidant enzymes thus making the cells more prone tooxidative stress [18] Suggested mechanism included directinhibition of NADP-dependent enzymes such as glucose 6-phosphate dehydrogenase glyceraldehyde 3-phosphate dehy-drogenase and lactate dehydrogenase In agreement withour data evidence from different group exists showing thatDPI is exerting prooxidative effects in given cell types andconditions [17] Further it was reported that APO contrary toDPI stimulates PPP which is known to be a subsequent stepfollowing oxidative stress exposure This was prevented byaddition of GSH into medium further suggesting that APO-induced GSH oxidation might be involved in observed PPPactivation [11] However in a different study APO opposinglyincreased the synthesis of the GSH through activation oftranscription factor AP-1 Notably levels of GSSG were notaltered implying that APO itself does not cause oxidativestress [61] Many studies are highlighting that APO is aprodrug that must be first metabolized through oxidation toits oligomeric form thus certain redox environment mustbe present in order to achieve full APO activation [62] nar-rowing its function preferably to cells with strong ROS pro-duction like stimulated endothelial cells [63] or professionalphagocytes [64] In compliance with these findings it wasdemonstrated that APO can act both as an antioxidant andas a prooxidant depending on the cell type and its oxidizingpotential [9 10] This is in agreement with our experimentsperformed on nonphagocytic low-level ROS cells where APOmight rather contribute to oxidative stress as demonstratedby live imaging Interestingly different modifications of APOare suggested for in vivo experiments [65] further revealingthe complexity of this issue

Critical features of mES pluripotency self-renewal andunlimited proliferation are predominantly exerted throughactions of cytokine LIF which is routinely added to theculture medium Binding of LIF to its receptor triggersthe activity of three major intracellular signaling cascadesJAKStat3 PI3KAkt and MAPKErk These pathways con-verge to regulate the gene expression pattern typical for mES[27] To clarify possible effects of APO and DPI on selectedsignaling pathways the modulation of Stat3 Akt and Erkactivation status was analyzed in mES cells Firstly mES wereserum and LIF depleted in order to increase cellular responseas cultivation in complete medium leads to a constantactivation of those pathways Stimulation by LIF and FBSwas employed to reactivate signaling [25 66] As expectedLIF addition induced preferably Stat3 response while FBScontaining growth factors and cytokines augmented Akt andErk signaling To further analyze effect of drugs on selectedkinases we examined their phosphorylation status also incomplete medium after 1-hour and 24-hour treatment toelucidate early changes and later cell response in signaling

pathways In agreement with other studies both Akt and Erkkinases responded in ROS-sensitive manner [67 68] whichwe demonstrated by H

2O2-induced phosphorylation Simi-

larly we can assume that DPI-mediated ROS production isresponsible for the observed effects on Akt and Erk activationwhich is in contrast to the response to APO Properties ofgeneral antioxidant NAC in sense of attenuation of kinasephosphorylation were more profound during treatment incomplete medium Notably we did not observe changes ofStat3 phosphorylation in presence of any drugs tested in ourstudy The most striking effect was detected downstream ofPI3K on the level of S473 Akt phosphorylation when APOtreatmentwas employed In serum starved cells in FBS or LIFactivated cells and also in the presence of complete mediumAPOdecreasedAkt phosphorylationwhen short term impactwas studied It was earlier reported that vanillin and someof its derivatives including APO readily inhibited PI3K inlung adenocarcinoma cell line [69] These authors are alsosuggesting in agreement with our data that radical scaveng-ing or other antioxidant properties of those compounds arenot responsible for the observed effect Thus the mode andmechanism of inhibition needs to be further clarified

Remarkably critical role of PI3K was also described forthe process of NOX activation [70] thus it can be hypoth-esized that some of the APO inhibitory actions towardsNOXs might be also mediated via PI3K inhibition Tofurther elucidate impact of APO-induced PI3K inhibitionwe examined the level of homeodomain transcription fac-tor Nanog which was suggested as a downstream targetof PI3K and is also recognized as an important intrinsicregulator of stem cell pluripotency [28] After 24 hours ofAPO treatment the level of Nanog was dramatically reducedInterestingly Akt phosphorylation was upregulated by APOafter 24 hours compared to untreated cells and this increasecould be reverted by addition of NAC although Nanoglevels remained diminished suggesting that this impairmentis perhaps not related to APO-induced modulation of ROSlevels in the cell Phosphorylation of both Akt and Erk wasaugmented by DPI treatment that may be attributed to itsabove described prooxidative effect as it was reduced byNACsupplementation in the case of Akt

Next we examined the impact of NOX inhibitors oncanonical Wnt signaling as it also plays a distinctive role inregulation of embryonic stem cells [31 71] and the modulatorof this pathway GSK3 is a known PI3K downstream [72]GSK3 regulates Wnt pathway by phosphorylating 120573-cateninon multiple sites that enhances its subsequent degradation[43] GSK3 is active in resting cells but is readily inhib-ited through the PI3KAkt-mediated phosphorylation of N-terminal serine residues (S9 in GSK3120573 and S21 in GSK3120572)[72] Therefore in the conventional view it is assumed thatactivity of PI3KAkt should promote canonical Wnt signal-ing In contrast to this experimental evidence argues againstthis simplified scenario as Akt activation failed to promoteWnt120573-catenin signaling when insulin and constitutivelyactive Akt were administrated [73] Moreover it was reportedthat Axin-associated GSK3 responsible for mediating 120573-catenin degradation represents just aminor fraction of GSK3cellular content and this complex is not accessible to Akt


phosphorylation thus preventing PI3KAktWnt cross-talk[74] In our experiments we did not see any effect onbasal Wnt transcription activity when NOX inhibitors or LYwas employed Moreover treatment of both APO and LYdecreased GSK3120573 S9 phosphorylation further questioningthe simplified model of PI3KWnt cross-talk mediated solelyby level of GSK3 inhibition However after Wnt3a inductionboth LY and APO augmented Wnt transcription activityNotably PI3K inhibition was recently shown to increasethe amount of active nuclear 120573-catenin and to promoteinduction in TOPflash assay in epithelial cell culture system[75] In agreement with other studies [76 77] authors weresuggesting the pivotal role of bidirectional loop betweenreceptor tyrosine kinase-driven MAPKs and Wnt120573-cateninsignaling Although Erk kinase was not dramatically affectedby APO treatment in our system we are not excluding thepossibility of a different MAPK involvement in observedphenomena as numerous convergence points were describedbetween PI3K and MAPKs [78]

5 Conclusions

Altogether our study suggests prooxidant activity of APOand DPI in mES Moreover treatment with those drugsresults in different modulation of intracellular pathways crit-ical for regulation of proliferation and differentiation APOmarkedly downregulates activity of Akt and its downstreamNanog and augments Wnt signaling DPI promotes Aktand Erk activation Taking into account described negligibleNOXs levels inmESwe suggest that actions of drugs observedin our experiments are rather independent of their typicalfunction as NOX inhibitors Therefore caution should betaken to potential applications of these NOX inhibitorsand interpretation of obtained results especially in studiesfocused on the stem cell biology and intracellular and redoxsignaling

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paperThe authors alone areresponsible for the content and writing of the paper

Acknowledgments

The authors would like to thank Dr V Bryja (Institute ofExperimental Biology Masaryk University) for a helpfulWntsignaling discussion Computational resourceswere providedby theMetaCentrum under the LM2010005 program and theCERIT-SC under the Centre CERIT Scientific Cloud Pro-gram part of the Research and Development for InnovationsOperational Program CZ1053200080144 The work wasfinancially supported by the European Regional Develop-ment FundmdashProject FNUSA-ICRC CZ1051100020123by the FP7 Regpot ICRC-ERA-HumanBridge No 316345funds from the Faculty of Medicine of Masaryk Univer-sity MUNIA15582014 and Ministry of Education Youthand Sport of the Czech Republic Grant MSM0021622430

This research is also supported by Collaboration betweenMasaryk University and Karolinska Institutet Project KI-MU Grant CZ1072300200180 and the Employment ofNewly Graduated Doctors of Science for Scientific ExcellenceProgram CZ1072300300009 cofinanced from EuropeanSocial Fund and the state budget of the Czech Republic

References

[1] M Valko D Leibfritz J Moncol M T D Cronin M Mazurand J Telser ldquoFree radicals and antioxidants in normal physi-ological functions and human diseaserdquo International Journal ofBiochemistry and Cell Biology vol 39 no 1 pp 44ndash84 2007

[2] W Droge ldquoFree radicals in the physiological control of cellfunctionrdquo Physiological Reviews vol 82 no 1 pp 47ndash95 2002

[3] S Dikalov ldquoCross talk between mitochondria and NADPHoxidasesrdquo Free Radical Biology and Medicine vol 51 no 7 pp1289ndash1301 2011

[4] J D Lambeth ldquoNOX enzymes and the biology of reactiveoxygenrdquo Nature Reviews Immunology vol 4 no 3 pp 181ndash1892004

[5] F Jiang Y Zhang and G J Dusting ldquoNADPH oxidase-mediated redox signaling roles in cellular stress response stresstolerance and tissue repairrdquo Pharmacological Reviews vol 63no 1 pp 218ndash242 2011

[6] K Bedard and K-H Krause ldquoThe NOX family of ROS-generatingNADPHoxidases physiology and pathophysiologyrdquoPhysiological Reviews vol 87 no 1 pp 245ndash313 2007

[7] S Altenhofer K A Radermacher PWM Kleikers KWinglerand H H H W Schmidt ldquoEvolution of NADPH oxidaseinhibitors selectivity and mechanisms for target engagementrdquoAntioxidantsampRedox Signaling vol 23 no 5 pp 406ndash427 2015

[8] J Stefanska and R Pawliczak ldquoApocynin molecular aptitudesrdquoMediators of Inflammation vol 2008 Article ID 106507 10pages 2008

[9] S Heumuller SWind E Barbosa-Sicard et al ldquoApocynin is notan inhibitor of vascular NADPH oxidases but an antioxidantrdquoHypertension vol 51 no 2 pp 211ndash217 2008

[10] M Vejrazka R Mıcek and S Stıpek ldquoApocynin inhibitsNADPH oxidase in phagocytes but stimulates ROS produc-tion in non-phagocytic cellsrdquo Biochimica et Biophysica ActamdashGeneral Subjects vol 1722 no 2 pp 143ndash147 2005

[11] C Riganti C Costamagna A Bosia and D Ghigo ldquoTheNADPH oxidase inhibitor apocynin (acetovanillone) inducesoxidative stressrdquo Toxicology and Applied Pharmacology vol 212no 3 pp 179ndash187 2006

[12] L R G Castor K A Locatelli and V F Ximenes ldquoPro-oxidantactivity of apocynin radicalrdquo Free Radical Biology andMedicinevol 48 no 12 pp 1636ndash1643 2010

[13] F Engels B F Renirie B A rsquoT Hart R P Labadie and F PNijkamp ldquoEffects of apocynin a drug isolated from the rootsof Picrorhiza kurroa on arachidonic acid metabolismrdquo FEBSLetters vol 305 no 3 pp 254ndash256 1992

[14] S Wind K Beuerlein T Eucker et al ldquoComparative phar-macology of chemically distinct NADPH oxidase inhibitorsrdquoBritish Journal of Pharmacology vol 161 no 4 pp 885ndash8982010

[15] V B OrsquoDonnell D G Tew O T G Jones and P J EnglandldquoStudies on the inhibitory mechanism of iodonium compounds


with special reference to neutrophil NADPHoxidaserdquoBiochem-ical Journal vol 290 part 1 pp 41ndash49 1993

[16] Y Li and M A Trush ldquoDiphenyleneiodonium an NAD(P)Hoxidase inhibitor also potently inhibits mitochondrial reac-tive oxygen species productionrdquo Biochemical and BiophysicalResearch Communications vol 253 no 2 pp 295ndash299 1998

[17] N Li K Ragheb G Lawler et al ldquoDPI induces mitochon-drial superoxide-mediated apoptosisrdquo Free Radical Biology andMedicine vol 34 no 4 pp 465ndash477 2003

[18] C Riganti E Gazzano M Polimeni C Costamagna ABosia and D Ghigo ldquoDiphenyleneiodonium inhibits the cellredox metabolism and induces oxidative stressrdquo The Journal ofBiological Chemistry vol 279 no 46 pp 47726ndash47731 2004

[19] E K Weir C N Wyatt H L Reeve J Huang S L Archer andC Peers ldquoDiphenyleneiodonium inhibits both potassium andcalcium currents in isolated pulmonary artery smooth musclecellsrdquo Journal of Applied Physiology vol 76 no 6 pp 2611ndash26151994

[20] K Wang T Zhang Q Dong E C Nice C Huang and Y WeildquoRedox homeostasis the linchpin in stem cell self-renewal anddifferentiationrdquo Cell Death amp Disease vol 4 no 3 article e5372013

[21] Y M Cho S Kwon Y K Pak et al ldquoDynamic changes inmitochondrial biogenesis and antioxidant enzymes during thespontaneous differentiation of human embryonic stem cellsrdquoBiochemical andBiophysical ResearchCommunications vol 348no 4 pp 1472ndash1478 2006

[22] H Sauer G Rahimi J Hescheler and M Wartenberg ldquoEffectsof electrical fields on cardiomyocyte differentiation of embry-onic stem cellsrdquo Journal of Cellular Biochemistry vol 75 no 4pp 710ndash723 1999

[23] E Serena E Figallo N Tandon et al ldquoElectrical stimulationof human embryonic stem cells cardiac differentiation andthe generation of reactive oxygen speciesrdquo Experimental CellResearch vol 315 no 20 pp 3611ndash3619 2009

[24] A-R Ji S-Y Ku M S Cho et al ldquoReactive oxygen spe-cies enhance differentiation of human embryonic stem cells intomesendodermal lineagerdquo Experimental and Molecular Medi-cine vol 42 no 3 pp 175ndash186 2010

[25] H Niwa T Burdon I Chambers and A Smith ldquoSelf-renewalof pluripotent embryonic stem cells is mediated via activationof STAT3rdquoGenes ampDevelopment vol 12 no 13 pp 2048ndash20601998

[26] L S Ling D Voskas and J R Woodgett ldquoActivation of PDK-1maintains mouse embryonic stem cell self-renewal in a PKB-dependent mannerrdquo Oncogene vol 32 no 47 pp 5397ndash54082013

[27] H Hirai P Karian and N Kikyo ldquoRegulation of embryonicstem cell self-renewal and pluripotency by leukaemia inhibitoryfactorrdquo Biochemical Journal vol 438 no 1 pp 11ndash23 2011

[28] M P Storm B Kumpfmueller BThompson et al ldquoCharacteri-zation of the phosphoinositide 3-kinase-dependent transcrip-tome in murine embryonic stem cells identification of novelregulators of pluripotencyrdquo Stem Cells vol 27 no 4 pp 764ndash775 2009

[29] H Kotasova I Vesela J Kucera et al ldquoPhosphoinositide 3-kinase inhibition enables retinoic acid-induced neurogenesis inmonolayer culture of embryonic stem cellsrdquo Journal of CellularBiochemistry vol 113 no 2 pp 563ndash570 2012

[30] T Burdon C Stracey I Chambers J Nichols and A SmithldquoSuppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cellsrdquo Developmental Biol-ogy vol 210 no 1 pp 30ndash43 1999

[31] TMiki S-Y Yasuda andMKahn ldquoWnt120573-catenin signaling inembryonic stem cell self-renewal and somatic cell reprogram-mingrdquo Stem Cell Reviews and Reports vol 7 no 4 pp 836ndash8462011

[32] A R Simon U Rai B L Fanburg and B H Cochran ldquoActiva-tion of the JAK-STAT pathway by reactive oxygen speciesrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol 275 no 6pp C1640ndashC1652 1998

[33] Y Funato T Michiue M Asashima and H Miki ldquoThethioredoxin-related redox-regulating protein nucleoredoxininhibits Wnt-beta-catenin signalling through dishevelledrdquoNature Cell Biology vol 8 no 5 pp 501ndash508 2006

[34] R Konopka M Hyzdrsquoalova L Kubala and J Pachernık ldquoNewluminescence-based approach to measurement of luciferasegene expression reporter activity and adenosine triphosphate-based determination of cell viabilityrdquo Folia Biologica vol 56 no2 pp 66ndash71 2010

[35] A ECarpenter T R JonesMR Lamprecht et al ldquoCellProfilerimage analysis software for identifying and quantifying cellphenotypesrdquo Genome Biology vol 7 no 10 article R100 2006

[36] M Stoter ANiederlein R Barsacchi FMeyenhofer H Brandland M Bickle ldquoCellProfiler and KNIME open source tools forhigh content screeningrdquoMethods inMolecular Biology vol 986pp 105ndash122 2013

[37] M R Berthold N Cebron F Dill et al ldquoKNIME the konstanzinformation minerrdquo in Data Analysis Machine Learning andApplications C Preisach H Burkhardt L Schmidt-Thiemeand R Decker Eds Springer Berlin Germany 2008

[38] J Zielonka J Vasquez-Vivar and B Kalyanaraman ldquoDetectionof 2-hydroxyethidium in cellular systems a unique markerproduct of superoxide andhydroethidinerdquoNature Protocols vol3 no 1 pp 8ndash21 2008

[39] H Zhao J Joseph H M Fales et al ldquoDetection and character-ization of the product of hydroethidine and intracellular super-oxide by HPLC and limitations of fluorescencerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 102 no 16 pp 5727ndash5732 2005

[40] HCai SDikalov KKGriendling andDGHarrison ldquoDetec-tion of reactive oxygen species and nitric oxide in vascular cellsand tissues comparison of sensitivity and specificityrdquo Methodsin molecular medicine vol 139 pp 293ndash311 2007

[41] R E A de Groot R S Ganji O Bernatik et al ldquoHuwe1-mediated ubiquitylation of dishevelled defines a negative feed-back loop in the Wnt signaling pathwayrdquo Science Signaling vol7 no 317 article ra26 2014

[42] V Korinek N Barker P J Morin et al ldquoConstitutive transcrip-tional activation by a beta-catenin-Tcf complex inAPCminusminus coloncarcinomardquo Science vol 275 no 5307 pp 1784ndash1787 1997

[43] T Hagena and A Vidal-Puigb ldquoCharacterisation of the phos-phorylation of 120573-catenin at the GSK-3 priming site Ser45rdquoBiochemical andBiophysical ResearchCommunications vol 294no 2 pp 324ndash328 2002

[44] H Sauer G Rahimi J Hescheler and M Wartenberg ldquoRoleof reactive oxygen species and phosphatidylinositol 3-kinase incardiomyocyte differentiation of embryonic stem cellsrdquo FEBSLetters vol 476 no 3 pp 218ndash223 2000


[45] M Buggisch B Ateghang C Ruhe et al ldquoStimulation ofES-cell-derived cardiomyogenesis and neonatal cardiac cellproliferation by reactive oxygen species and NADPH oxidaserdquoJournal of Cell Science vol 120 part 5 pp 885ndash894 2007

[46] Q Xiao Z Luo A E Pepe A Margariti L Zeng and QXu ldquoEmbryonic stem cell differentiation into smooth musclecells is mediated by Nox4-produced H

2O2rdquo American Journal

of PhysiologymdashCell Physiology vol 296 no 4 pp C711ndashC7232009

[47] J Li M Stouffs L Serrander et al ldquoThe NADPH oxidaseNOX4 drives cardiac differentiation role in regulating cardiactranscription factors and MAP kinase activationrdquo MolecularBiology of the Cell vol 17 no 9 pp 3978ndash3988 2006

[48] F Chen S Haigh S Barman and D J R Fulton ldquoFrom formto function the role of Nox4 in the cardiovascular systemrdquoFrontiers in Physiology vol 3 article 412 2012

[49] L Serrander L Cartier K Bedard et al ldquoNOX4 activity isdetermined by mRNA levels and reveals a unique pattern ofROS generationrdquo Biochemical Journal vol 406 no 1 pp 105ndash114 2007

[50] J Rehman ldquoEmpowering self-renewal and differentiation therole of mitochondria in stem cellsrdquo Journal of MolecularMedicine vol 88 no 10 pp 981ndash986 2010

[51] S Sart L Song and Y Li ldquoControlling redox status for stem cellsurvival expansion and differentiationrdquo Oxidative Medicineand Cellular Longevity vol 2015 Article ID 105135 14 pages2015

[52] C Riganti C Costamagna S Doublier et al ldquoThe NADPHoxidase inhibitor apocynin induces nitric oxide synthesis viaoxidative stressrdquoToxicology andApplied Pharmacology vol 228no 3 pp 277ndash285 2008

[53] E Aldieri C Riganti M Polimeni et al ldquoClassical inhibitorsof NOX NAD(P)H oxidases are not specificrdquo Current DrugMetabolism vol 9 no 8 pp 686ndash696 2008

[54] M R Abid Z Kachra K C Spokes and W C Aird ldquoNADPHoxidase activity is required for endothelial cell proliferation andmigrationrdquo FEBS Letters vol 486 no 3 pp 252ndash256 2000

[55] R M Scaife ldquoSelective and irreversible cell cycle inhibition bydiphenyleneiodoniumrdquo Molecular Cancer Therapeutics vol 4no 6 pp 876ndash884 2005

[56] MYamasakiM Iwase KKawano Y SakakibaraM Suiko andK Nishiyama ldquoSelective inhibition by apocynin of the prolifer-ation and adhesion to fibronectin of v-H-ras-transformed 3Y1cellsrdquo Bioscience Biotechnology and Biochemistry vol 76 no 6pp 1177ndash1181 2012

[57] S Suzuki K Shiraga S Sato et al ldquoApocynin an NADPH oxi-dase inhibitor suppresses rat prostate carcinogenesisrdquo CancerScience vol 104 no 12 pp 1711ndash1717 2013

[58] S Suzuki P Pitchakarn S Sato T Shirai and S TakahashildquoApocynin an NADPH oxidase inhibitor suppresses progres-sion of prostate cancer via Rac1 dephosphorylationrdquo Experi-mental and Toxicologic Pathology vol 65 no 7-8 pp 1035ndash10412013

[59] B Fink K Laude LMcCann A Doughan D G Harrison andS Dikalov ldquoDetection of intracellular superoxide formation inendothelial cells and intact tissues using dihydroethidium andan HPLC-based assayrdquo American Journal of Physiology - CellPhysiology vol 287 no 4 pp C895ndashC902 2004

[60] B Kalyanaraman B P Dranka M Hardy R Michalski andJ Zielonka ldquoHPLC-based monitoring of products formed

from hydroethidine-based fluorogenic probesmdashthe ultimateapproach for intra- and extracellular superoxide detectionrdquoBiochimica et BiophysicaActa vol 1840 no 2 pp 739ndash744 2014

[61] T S Lapperre L A Jimenez F Antonicelli et al ldquoApocyninincreases glutathione synthesis and activates AP-1 in alveolarepithelial cellsrdquo FEBS Letters vol 443 no 2 pp 235ndash239 1999

[62] V F XimenesM P P Kanegae S R Rissato andM SGalhianeldquoThe oxidation of apocynin catalyzed by myeloperoxidase pro-posal for NADPH oxidase inhibitionrdquo Archives of Biochemistryand Biophysics vol 457 no 2 pp 134ndash141 2007

[63] D K Johnson K J Schillinger D M Kwait et al ldquoInhi-bition of NADPH oxidase activation in endothelial cells byortho-methoxy-substituted catecholsrdquo Endothelium Journal ofEndothelial Cell Research vol 9 no 3 pp 191ndash203 2002

[64] J Stolk T J Hiltermann J H Dijkman and A J VerhoevenldquoCharacteristics of the inhibition of NADPH oxidase activationin neutrophils by apocynin a methoxy-substituted catecholrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol11 no 1 pp 95ndash102 1994

[65] Q Wang R E Smith R Luchtefeld et al ldquoBioavailability ofapocynin through its conversion to glycoconjugate but not todiapocyninrdquo Phytomedicine vol 15 no 6-7 pp 496ndash503 2008

[66] M Abkhezr A R Keramati S N Ostad J Davoodi and MH Ghahremani ldquoThe time course of Akt and ERK activationon XIAP expression in HEK 293 cell linerdquo Molecular BiologyReports vol 37 no 4 pp 2037ndash2042 2010

[67] M Torres and H J Forman ldquoRedox signaling and the MAPkinase pathwaysrdquo BioFactors vol 17 no 1ndash4 pp 287ndash296 2003

[68] D Trachootham W Lu M A Ogasawara N R-D Valle andP Huang ldquoRedox regulation of cell survivalrdquo Antioxidants ampRedox Signaling vol 10 no 8 pp 1343ndash1374 2008

[69] K Lirdprapamongkol J-P Kramb T Suthiphongchai et alldquoVanillin suppresses metastatic potential of human cancer cellsthrough PI3K inhibition and decreases angiogenesis in VivordquoJournal of Agricultural and Food Chemistry vol 57 no 8 pp3055ndash3063 2009

[70] S Chatterjee E A Browning N Hong et al ldquoMembranedepolarization is the trigger for PI3KAkt activation and leadsto the generation of ROSrdquoThe American Journal of PhysiologyHeart and Circulatory Physiology vol 302 no 1 pp H105ndashH1142012

[71] N Sato L Meijer L Skaltsounis P Greengard and A HBrivanlou ldquoMaintenance of pluripotency in human and mouseembryonic stem cells through activation of Wnt signaling bya pharmacological GSK-3-specific inhibitorrdquo Nature Medicinevol 10 no 1 pp 55ndash63 2004

[72] D A E Cross D R Alessi P Cohen M Andjelkovich andB A Hemmings ldquoInhibition of glycogen synthase kinase-3 byinsulinmediated by protein kinase BrdquoNature vol 378 no 6559pp 785ndash789 1995

[73] D P Brazil J Park and B A Hemmings ldquoPKB bindingproteinsrdquo Cell vol 111 no 3 pp 293ndash303 2002

[74] S S Ng T Mahmoudi E Danenberg et al ldquoPhosphatidylinos-itol 3-kinase signaling does not activate the Wnt cascaderdquo TheJournal of Biological Chemistry vol 284 no 51 pp 35308ndash353132009

[75] N T Georgopoulos L A Kirkwood and J Southgate ldquoA novelbidirectional positive-feedback loop between Wnt-120573-cateninand EGFR-ERK plays a role in context-specific modulation ofepithelial tissue regenerationrdquo Journal of Cell Science vol 127part 13 pp 2967ndash2982 2014


[76] I Cervenka J Wolf J Masek et al ldquoMitogen-activated proteinkinases promote WNT120573-catenin signaling via phosphoryla-tion of LRP6rdquoMolecular and Cellular Biology vol 31 no 1 pp179ndash189 2011

[77] P Krejci A AklianM Kaucka et al ldquoReceptor tyrosine kinasesactivate canonical WNT120573-catenin signaling via MAP kinaseLRP6 pathway and direct 120573-catenin phosphorylationrdquo PLoSONE vol 7 no 4 Article ID e35826 2012

[78] M C Mendoza E E Er and J Blenis ldquoThe Ras-ERK andPI3K-mTORpathways cross-talk and compensationrdquoTrends inBiochemical Sciences vol 36 no 6 pp 320ndash328 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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EndocrinologyInternational Journal of



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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


reliably block particular NOX The most frequently usedinhibitors employed in experiments are vanillin derivative 4-hydroxy-3-methoxyacetophenone (trivial names apocyninor acetovanillone APO) and diphenyleneiodonium chloride(DPI) Both of these drugs were applied in numerous in vitroand in vivo studies and although their effect was attributedprimarily to NOX inhibition specificity of APO and DPIremains questioned [7]

The proposed molecular mechanism of APO-mediatedNOX inhibition is not fully understood but it involves impair-ment of NOX complex assembly and activation [8] APO wasalso shown to act directly as ROS scavenger [9] Contrarily tothis finding other studies suggest that APO is rather a proox-idant stimulating ROS production [10ndash12] Further APO wasalso shown to modulate the generation of arachidonic acid-derived inflammatory mediators [13]

DPI was reported not only to affect NOX but also tointerfere with other flavoenzymes including nitric oxidesynthase and xanthine oxidase [14 15] Inhibitory effects ofDPI onmitochondrial ROS production were also shown [16]On the other hand DPI induces O

2

∙minus mediated apoptosis[17] inhibits cell redox metabolism and promotes generaloxidative stress [18] DPI is also suggested as a nonselectiveblocker of ionic channels [19] The above mentioned nonspe-cific effects are thought to be responsible for contradictoryresults obtained using these inhibitors Further studies areneeded to better understand the actions of APO and DPI notdirectly related to NOX inhibition

Intracellular formation of ROS leading to overall redoxstatus modulation is important for regulation of pluripotentcells differentiation [20] In mouse embryonic stem cells(mES) pluripotent cells derived from inner cell mass ofblastocyst redox alterations are thought to play a role in thebalance between self-renewal anddifferentiationUndifferen-tiated mES have several times lower ROS level in comparisonwith differentiating mES [21] It was shown that short termincrease of the ROS favors differentiation into cardiomy-ocytes [22 23] and into endoderm and mesoderm lineage[24]

Several signaling pathways are crucial for the regulationof self-renewal and differentiation ofmES Primarily themESpluripotency is controlled by Stat3 together with PI3KAktsignaling pathways [25ndash27] Importance of PI3K signalingwas demonstrated in many studies where its inhibitionnegatively regulates the self-renewal inmES [28 29] FurtherMAPKErk signaling pathway is rather important for mESdifferentiation as its inhibition improves maintenance ofthe pluripotent stem cell phenotype [30] A growing bodyof evidence indicates that Wnt120573-catenin signaling pathwayplays a vital role in the regulation of mES fate [31] Signalingpathways including Stat3 PI3KAkt Erk and Wnt are mod-ified by ROS production [5 32 33] and therefore might beaffected by NOX or other ROS modulating agents althoughthe importance of this phenomenon in mES remains elusive

Our study demonstrates unexpected prooxidant activityof APO and DPI in undifferentiated mES together withimpairment of cell proliferation Further we show that APOinhibits PI3K signaling accompanied by decrease in proteinlevel of critical ES pluripotency regulator Nanog whereas

DPI enhances both PI3K and Erk activity Interestingly APOstrengthens Wnt activity pointing out another unknownmechanism of APO-mediated changes in signaling cascadesregulating mES In contrast to PI3K and Erk we did notobserve any effect of APO or DPI on Stat3 phosphorylationwhich is considered to play the major role in mES mainte-nance

2 Material and Methods

21 Cell Culture and Treatment A feeder-free adapted mESline R1 was propagated as described previously [29] in anundifferentiated state by cell culturing on tissue cultureplastic coated by gelatin (01 porcine gelatin solution inwater) in Dulbeccorsquos modified Eaglersquos medium (DMEM)containing 15 fetal bovine serum (FBS) 100 IUmL peni-cillin 01mgmL streptomycin 1x nonessential amino acid(all from Gibco-Invitrogen UK) and 005mM 120573-mercap-toethanol (Sigma-Aldrich USA) supplemented with 5 ngmL of leukemia inhibitory factor (LIF Chemicon USA) re-ferred to here as the complete medium

APO DPI hydrogen peroxide (H2O2) N-acetylcysteine

(NAC) and LY294002 (LY) were provided from Sigma-Aldrich Stock solutions of APO (04M) DPI (10mM) andLY (10mM) were prepared by dissolving the compounds indimethyl sulfoxide Aliquots were stored at minus20∘C NAC wasprepared as a 05M stock solution in serum-free DMEMmedium pH was adjusted to 74 and filter-sterilized aliquotswere stored at minus20∘C Drugs were added directly to the incu-bation medium or freshly prediluted in sterile phosphate-buffered saline (PBS) to desired concentration

22 Cell Proliferation The cell proliferation was determinedby estimation of overall cellular protein mass in wholecell lysates that reflects the cell number as demonstratedpreviously [34] ES cells were seeded to 24-well plate incomplete media at density 5 000 cells per cm2 Next day thecells were treated with drugs for further 48 hours Finally thecells were washed twice with PBS and lysed in SDS buffer(50mM Tris-HCl pH 75 100mM NaCl 10 glycerol 1SDS 1mM EDTA) Protein concentration was determinedusing DC protein assay (Bio-Rad USA) kit according tomanufacturerrsquos instructions

23 Determination of NOX Expression Total RNA wasextracted using the UltraClean Tissue amp Cells RNA IsolationKit (MO BIO Laboratories USA) for ESC and RNAzol RT(Molecular research center Inc USA) for mouse tissuesaccording to manufacturerrsquos instructions 1 120583g of total RNAwas used for cDNA synthesis with DyNAmo cDNA Syn-thesis Kit (Finnzymes Finland) according to the manufac-turerrsquos instructions qPCR was performed in LightCycler 480instrument using LightCycler Probes Master with UniversalProbe library probes (all from Roche Germany) accord-ing to manufacturerrsquos instructions Ribosomal protein L13A(RPL13A) was used as reference gene data are presented as








2

∙minus



2OCH

3CN Kromasil C18





3 Results




NOX1

Heart mES00000

00002

00004

00006

00008Re

lativ

e mRN

A ex

pres

sion

sim20x

(a)

NOX2

Spleen mES000

001

002

003

004

005

Relat

ive m

RNA

expr

essio

n

sim900x

(b)

NOX3

Kidney mES0000

0002

0004

0006

0008

0010

Relat

ive m

RNA

expr

essio

n

sim300x

(c)

NOX4

Kidney mES00

01

02

03

04

05Re

lativ

e mRN

A ex

pres

sion

sim850x

(d)

DUOX1

Lung mES00000

00005

00010

00015

Relat

ive m

RNA

expr

essio

n

sim7x

(e)

DUOX2

Lung mES00000

00002

00004

00006

00008

Relat

ive m

RNA

expr

essio

n

sim60x

(f)

Figure 1 Continued


NOXs in mES


00002

00004

00006

Relat

ive m

RNA

expr

essio

n

(g)


Cel

l gro

wth

( o

f ctr

)

150

100

50

0

Ctr 01 025 05 1 2

A AA

B

C

D

APO

(mM)

(a)

Cel

l gro

wth

( o

f ctr

)150

100

50

0

AA

B

BC BC BC

C

DPI

Ctr 100 200 400 800 1600 3200

(nM)

(b)




2



2





5

4

3

2

1

0

0 20 40 60 80 100 120

Relat

ive fl

uore

scen

ce

(minutes)

CtrAPO

(a)

5

4

3

2

1

0

Relat

ive fl

uore

scen

ce

0 20 40 60 80 100 120

(minutes)

CtrDPI

(b)

3

2

1

0

Relat

ive fl

uore

scen

ce

APOCtr DPI

lowastlowast

(c)APOCtr DPI

15

10

05

00

Relat

ive u

nits

(are

a of p

eak)

2-OH-E(+)

(d)

APOCtr DPI

lowast

15

10

05

00

Relat

ive u

nits

(are

a of p

eak)

25

20

E(+)

(e)



Stat3

p-Stat3

p-Akt

p-Erk

Akt

Erk

LIF FBSSF


120573-actin














4 Discussion





p-Akt

p-Erk

Akt

Erk

Ctr APO DPI NAC

120573-actin

H2O2

(a)

p-Akt

p-Erk

Akt

Erk

Ctr APO DPI NAC

120573-actin

H2O2

(b)



NAC

p-Akt

Akt

p-Erk

Erk

Nanog


120573-actin







0

20

40

60Re

lativ

e lum

ines

cenc

e


lowastlowast

lowastlowastlowast

(a)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(b)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI

lowastlowast

(c)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(d)

Wnt3a

Ctr

LY APO

DPI

Ctr

LY APO

DPI

Ctr

LY APO

DPI

S33-120573-catenin

p-GSK3120573

120573-actin

(e)



2



2




2











5 Conclusions




Acknowledgments



References



























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























2

∙minus



2OCH

3CN Kromasil C18





3 Results




NOX1

Heart mES00000

00002

00004

00006

00008Re

lativ

e mRN

A ex

pres

sion

sim20x

(a)

NOX2

Spleen mES000

001

002

003

004

005

Relat

ive m

RNA

expr

essio

n

sim900x

(b)

NOX3

Kidney mES0000

0002

0004

0006

0008

0010

Relat

ive m

RNA

expr

essio

n

sim300x

(c)

NOX4

Kidney mES00

01

02

03

04

05Re

lativ

e mRN

A ex

pres

sion

sim850x

(d)

DUOX1

Lung mES00000

00005

00010

00015

Relat

ive m

RNA

expr

essio

n

sim7x

(e)

DUOX2

Lung mES00000

00002

00004

00006

00008

Relat

ive m

RNA

expr

essio

n

sim60x

(f)

Figure 1 Continued


NOXs in mES


00002

00004

00006

Relat

ive m

RNA

expr

essio

n

(g)


Cel

l gro

wth

( o

f ctr

)

150

100

50

0

Ctr 01 025 05 1 2

A AA

B

C

D

APO

(mM)

(a)

Cel

l gro

wth

( o

f ctr

)150

100

50

0

AA

B

BC BC BC

C

DPI

Ctr 100 200 400 800 1600 3200

(nM)

(b)




2



2





5

4

3

2

1

0

0 20 40 60 80 100 120

Relat

ive fl

uore

scen

ce

(minutes)

CtrAPO

(a)

5

4

3

2

1

0

Relat

ive fl

uore

scen

ce

0 20 40 60 80 100 120

(minutes)

CtrDPI

(b)

3

2

1

0

Relat

ive fl

uore

scen

ce

APOCtr DPI

lowastlowast

(c)APOCtr DPI

15

10

05

00

Relat

ive u

nits

(are

a of p

eak)

2-OH-E(+)

(d)

APOCtr DPI

lowast

15

10

05

00

Relat

ive u

nits

(are

a of p

eak)

25

20

E(+)

(e)



Stat3

p-Stat3

p-Akt

p-Erk

Akt

Erk

LIF FBSSF


120573-actin














4 Discussion





p-Akt

p-Erk

Akt

Erk

Ctr APO DPI NAC

120573-actin

H2O2

(a)

p-Akt

p-Erk

Akt

Erk

Ctr APO DPI NAC

120573-actin

H2O2

(b)



NAC

p-Akt

Akt

p-Erk

Erk

Nanog


120573-actin







0

20

40

60Re

lativ

e lum

ines

cenc

e


lowastlowast

lowastlowastlowast

(a)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(b)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI

lowastlowast

(c)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(d)

Wnt3a

Ctr

LY APO

DPI

Ctr

LY APO

DPI

Ctr

LY APO

DPI

S33-120573-catenin

p-GSK3120573

120573-actin

(e)



2



2




2











5 Conclusions




Acknowledgments



References



























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NOX1

Heart mES00000

00002

00004

00006

00008Re

lativ

e mRN

A ex

pres

sion

sim20x

(a)

NOX2

Spleen mES000

001

002

003

004

005

Relat

ive m

RNA

expr

essio

n

sim900x

(b)

NOX3

Kidney mES0000

0002

0004

0006

0008

0010

Relat

ive m

RNA

expr

essio

n

sim300x

(c)

NOX4

Kidney mES00

01

02

03

04

05Re

lativ

e mRN

A ex

pres

sion

sim850x

(d)

DUOX1

Lung mES00000

00005

00010

00015

Relat

ive m

RNA

expr

essio

n

sim7x

(e)

DUOX2

Lung mES00000

00002

00004

00006

00008

Relat

ive m

RNA

expr

essio

n

sim60x

(f)

Figure 1 Continued


NOXs in mES


00002

00004

00006

Relat

ive m

RNA

expr

essio

n

(g)


Cel

l gro

wth

( o

f ctr

)

150

100

50

0

Ctr 01 025 05 1 2

A AA

B

C

D

APO

(mM)

(a)

Cel

l gro

wth

( o

f ctr

)150

100

50

0

AA

B

BC BC BC

C

DPI

Ctr 100 200 400 800 1600 3200

(nM)

(b)




2



2





5

4

3

2

1

0

0 20 40 60 80 100 120

Relat

ive fl

uore

scen

ce

(minutes)

CtrAPO

(a)

5

4

3

2

1

0

Relat

ive fl

uore

scen

ce

0 20 40 60 80 100 120

(minutes)

CtrDPI

(b)

3

2

1

0

Relat

ive fl

uore

scen

ce

APOCtr DPI

lowastlowast

(c)APOCtr DPI

15

10

05

00

Relat

ive u

nits

(are

a of p

eak)

2-OH-E(+)

(d)

APOCtr DPI

lowast

15

10

05

00

Relat

ive u

nits

(are

a of p

eak)

25

20

E(+)

(e)



Stat3

p-Stat3

p-Akt

p-Erk

Akt

Erk

LIF FBSSF


120573-actin














4 Discussion





p-Akt

p-Erk

Akt

Erk

Ctr APO DPI NAC

120573-actin

H2O2

(a)

p-Akt

p-Erk

Akt

Erk

Ctr APO DPI NAC

120573-actin

H2O2

(b)



NAC

p-Akt

Akt

p-Erk

Erk

Nanog


120573-actin







0

20

40

60Re

lativ

e lum

ines

cenc

e


lowastlowast

lowastlowastlowast

(a)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(b)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI

lowastlowast

(c)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(d)

Wnt3a

Ctr

LY APO

DPI

Ctr

LY APO

DPI

Ctr

LY APO

DPI

S33-120573-catenin

p-GSK3120573

120573-actin

(e)



2



2




2











5 Conclusions




Acknowledgments



References



























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NOXs in mES


00002

00004

00006

Relat

ive m

RNA

expr

essio

n

(g)


Cel

l gro

wth

( o

f ctr

)

150

100

50

0

Ctr 01 025 05 1 2

A AA

B

C

D

APO

(mM)

(a)

Cel

l gro

wth

( o

f ctr

)150

100

50

0

AA

B

BC BC BC

C

DPI

Ctr 100 200 400 800 1600 3200

(nM)

(b)




2



2





5

4

3

2

1

0

0 20 40 60 80 100 120

Relat

ive fl

uore

scen

ce

(minutes)

CtrAPO

(a)

5

4

3

2

1

0

Relat

ive fl

uore

scen

ce

0 20 40 60 80 100 120

(minutes)

CtrDPI

(b)

3

2

1

0

Relat

ive fl

uore

scen

ce

APOCtr DPI

lowastlowast

(c)APOCtr DPI

15

10

05

00

Relat

ive u

nits

(are

a of p

eak)

2-OH-E(+)

(d)

APOCtr DPI

lowast

15

10

05

00

Relat

ive u

nits

(are

a of p

eak)

25

20

E(+)

(e)



Stat3

p-Stat3

p-Akt

p-Erk

Akt

Erk

LIF FBSSF


120573-actin














4 Discussion





p-Akt

p-Erk

Akt

Erk

Ctr APO DPI NAC

120573-actin

H2O2

(a)

p-Akt

p-Erk

Akt

Erk

Ctr APO DPI NAC

120573-actin

H2O2

(b)



NAC

p-Akt

Akt

p-Erk

Erk

Nanog


120573-actin







0

20

40

60Re

lativ

e lum

ines

cenc

e


lowastlowast

lowastlowastlowast

(a)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(b)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI

lowastlowast

(c)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(d)

Wnt3a

Ctr

LY APO

DPI

Ctr

LY APO

DPI

Ctr

LY APO

DPI

S33-120573-catenin

p-GSK3120573

120573-actin

(e)



2



2




2











5 Conclusions




Acknowledgments



References



























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















5

4

3

2

1

0

0 20 40 60 80 100 120

Relat

ive fl

uore

scen

ce

(minutes)

CtrAPO

(a)

5

4

3

2

1

0

Relat

ive fl

uore

scen

ce

0 20 40 60 80 100 120

(minutes)

CtrDPI

(b)

3

2

1

0

Relat

ive fl

uore

scen

ce

APOCtr DPI

lowastlowast

(c)APOCtr DPI

15

10

05

00

Relat

ive u

nits

(are

a of p

eak)

2-OH-E(+)

(d)

APOCtr DPI

lowast

15

10

05

00

Relat

ive u

nits

(are

a of p

eak)

25

20

E(+)

(e)



Stat3

p-Stat3

p-Akt

p-Erk

Akt

Erk

LIF FBSSF


120573-actin














4 Discussion





p-Akt

p-Erk

Akt

Erk

Ctr APO DPI NAC

120573-actin

H2O2

(a)

p-Akt

p-Erk

Akt

Erk

Ctr APO DPI NAC

120573-actin

H2O2

(b)



NAC

p-Akt

Akt

p-Erk

Erk

Nanog


120573-actin







0

20

40

60Re

lativ

e lum

ines

cenc

e


lowastlowast

lowastlowastlowast

(a)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(b)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI

lowastlowast

(c)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(d)

Wnt3a

Ctr

LY APO

DPI

Ctr

LY APO

DPI

Ctr

LY APO

DPI

S33-120573-catenin

p-GSK3120573

120573-actin

(e)



2



2




2











5 Conclusions




Acknowledgments



References



























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Stat3

p-Stat3

p-Akt

p-Erk

Akt

Erk

LIF FBSSF


120573-actin














4 Discussion





p-Akt

p-Erk

Akt

Erk

Ctr APO DPI NAC

120573-actin

H2O2

(a)

p-Akt

p-Erk

Akt

Erk

Ctr APO DPI NAC

120573-actin

H2O2

(b)



NAC

p-Akt

Akt

p-Erk

Erk

Nanog


120573-actin







0

20

40

60Re

lativ

e lum

ines

cenc

e


lowastlowast

lowastlowastlowast

(a)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(b)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI

lowastlowast

(c)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(d)

Wnt3a

Ctr

LY APO

DPI

Ctr

LY APO

DPI

Ctr

LY APO

DPI

S33-120573-catenin

p-GSK3120573

120573-actin

(e)



2



2




2











5 Conclusions




Acknowledgments



References



























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















p-Akt

p-Erk

Akt

Erk

Ctr APO DPI NAC

120573-actin

H2O2

(a)

p-Akt

p-Erk

Akt

Erk

Ctr APO DPI NAC

120573-actin

H2O2

(b)



NAC

p-Akt

Akt

p-Erk

Erk

Nanog


120573-actin







0

20

40

60Re

lativ

e lum

ines

cenc

e


lowastlowast

lowastlowastlowast

(a)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(b)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI

lowastlowast

(c)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(d)

Wnt3a

Ctr

LY APO

DPI

Ctr

LY APO

DPI

Ctr

LY APO

DPI

S33-120573-catenin

p-GSK3120573

120573-actin

(e)



2



2




2











5 Conclusions




Acknowledgments



References



























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60Re

lativ

e lum

ines

cenc

e


lowastlowast

lowastlowastlowast

(a)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(b)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI

lowastlowast

(c)

00

05

10

15

20

Relat

ive l

umin

esce

nce

Ctr LY APO DPI(d)

Wnt3a

Ctr

LY APO

DPI

Ctr

LY APO

DPI

Ctr

LY APO

DPI

S33-120573-catenin

p-GSK3120573

120573-actin

(e)



2



2




2











5 Conclusions




Acknowledgments



References



























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2











5 Conclusions




Acknowledgments



References



























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusions




Acknowledgments



References



























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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