The Prostate 70:1119 ^1126 (2010)

Phorbol Ester Phorbol-12-Myristate-13-AcetateInduces EpithelialtoMesenchymal Transition in

HumanProstateCancerARCaPECells

Hui He,1 Alec J. Davidson,2 Daqing Wu,3 Fray F. Marshall,3

Leland W.K. Chung,3 Haiyen E. Zhau,3 Dalin He,1 and Ruoxiang Wang3*1DepartmentofUrology,The First Aff|liatedHospital, Xi’an JiaotongUniversity, Xi’an,China

2Neuroscience Institute,Morehouse SchoolofMedicine, Atlanta,Georgia3MolecularUrologyandTherapeutics,DepartmentofUrology,WinshipCancer Institute,

EmoryUniversity SchoolofMedicine, Atlanta,Georgia

BACKGROUND. We have reported that human prostate cancer ARCaPE cells undertakeepithelial to mesenchymal transition (EMT) when stimulated by certain soluble factors, andthat EMT is regulated by surface receptor-elicited signaling pathways through proteinphosphorylation. It is known that phorbol ester phorbol-12-myristate-13-acetate (PMA), apotent antagonist to both conventional and novel protein kinase C (PKC) isoenzymes, inducescancer cell scattering.METHODS. To assess the effect of PMA on EMT, ARCaPE cells were treated with PMA andwere assayed for EMT-related morphologic and behavioral changes. Specific inhibitors wereused to investigate the PMA-induced EMT.RESULTS. PMA at 100 nM induced EMT in a time-dependent manner, resulting in a completechange from epithelial to mesenchymal stromal morphology. Concurrently, PMA inhibitedexpression of epithelial marker E-cadherin and increased the level of stromal marker proteinvimentin, while the treated cells showed increased migratory and invasive capacities. Usingspecific inhibitors, we confirmed that the effect of PMA was mediated by PKC, whileisoenzymes of the novel PKC subfamily were implicated as the main mediator. Finally, wedetermined that the EMT was dependent on newly synthesized proteins, because inhibitors forgene transcription and protein translation could both inhibit the initiation of EMT.CONCLUSIONS. Although PMA is well known for its effects on cell migration and tumor for-mation, this work is the first to define PMA as an EMT inducer in prostate cancer cells. Furtherinvestigation in this experimental model may reveal important regulatory mechanisms and addi-tional molecular changes underlying EMT. Prostate 70: 1119–1126, 2010. # 2010 Wiley-Liss, Inc.

KEY WORDS: epithelial to mesenchymal transition; phorbol-12-myristate-13-acetate;protein kinase C; prostate cancer progression; metastasis

INTRODUCTION

Epithelial to mesenchymal transition (EMT) is afundamental process for epithelial cell remodelingduring embryonic development [1–4]. EMT is accom-panied by down-regulation of E-cadherin (E-cad), a keysurface protein for intercellular junction betweenepithelial cells, and by a switch of intermediate filamentprotein expression from cytokeratins to vimentin [3,4].Following EMT, epithelial cells adopt the morphologyand behavior of mesenchymal stromal cells, losingcellular polarity and becoming motile and invasive so

these cells can migrate to a specified site for furtherdevelopment and specialization. Through sequentialEMT events, epithelial cells of the primary ectodermform endoderm and mesoderm, which reorganize into

*Correspondence to: Ruoxiang Wang, Department of Urology,Emory University School of Medicine, 1365B Clifton Road, NE,Suite B5103, Atlanta, GA 30322. E-mail: rwang2@emory.eduReceived 5 January 2010; Accepted 20 January 2010DOI 10.1002/pros.21146Published online 23 March 2010 in Wiley InterScience(www.interscience.wiley.com).

+ 2010 Wiley-Liss, Inc.

epithelial cells for further EMT to form various somaticand internal visceral organs. After completion ofembryonic development and in adult stages, epithelialcells are considered to be in a stable state [5]. AdditionalEMT would cause migration and invasion of the epithe-lial cells into the mesenchymal stromal compartment.

The biological mechanism of EMT may be hijackedby prostate cancer cells, to the advantage of migrationand invasion [6–8]. Although cancer cells in thetransition state of an EMT are rarely seen and bonafide EMT in tumor specimens is controversial [9],prostate tumor cells are known to have the tendency tolose epithelial properties and acquire stromal charac-teristics. Similar to EMT in early embryogenesis, forexample, prostate cancer cells are frequently seen tohave lost E-cad expression [10], and to have switchedexpression of the intermediate filament protein expres-sion from cytokeratins to vimentin [11], while loss ofpolarity and acquired motility are common featuresof prostate tumor cells. Moreover, the EMT-likephenotype is positively correlated to prostate cancerprogression and metastasis [6], making EMT a highlyrelevant issue to prostate cancer progression andmetastasis.

We used human prostate cancer ARCaPE cells tostudy the mechanism of EMT during prostate cancerprogression and metastasis [6,8,12]. With an epithelialmorphology and tight intercellular junction, ARCaPE

cells form a cobblestone-like organization. Uponinduction by soluble factors, such as epidermal growthfactor (EGF), insulin-like growth factor (IGF-1), trans-forming growth factor (TGFb) [12,13], and b2-micro-globulin [6], these cells undertook a series ofmorphologic and behavioral changes reminiscent ofEMT. Upon overexpression of EMT-related genes, suchas the SNAIL [12], ZEB1 [13], and LIV-1 [6], ARCaPE

cells adopted the morphology of mesenchymal stromalcells with increased tumorigenic potency. When sub-jected to repeated inoculation in athymic mice, ARCaPE

cells recovered from xenograft tumors showed mesen-chymal stromal morphology and markedly increasedtumorigenic potential [8,14]. ARCaPE is an ideal modelfor studying abnormal EMT or EMT-like changesduring prostate progression and metastasis [6].

The mechanism regulating EMT-like changes inprostate cancer progression and metastasis remains tobe elucidated. EMT in ARCaPE cells could be inducedby soluble factors [12,13], probably through surfacereceptor-mediated intracellular signaling, while intra-cellular signal transduction pathway critical to the EMThas yet to be identified. On the other hand, it is wellknown that specific chemical reagents can functionas second messengers to activate specific signal trans-duction pathways. These reagents are useful tools foridentifying critical signal transduction mediators.

Employing these reagents, we examined signal trans-duction pathways for their role in promoting EMTin ARCaPE cells. In this report using behavioral andexpressional assays, we identified phorbol ester,phorbol-12-myristate-13-acetate (PMA), a specificagonist of the protein kinase C (PKC) isoenzymes, asa potent inducer of EMT-like changes in ARCaPE cells.

MATERIALSANDMETHODS

Reagents

PMA, Go6983, cyclohexamide (CHX), and actino-mycin D (Act.D) were purchased from Sigma–Aldrich(St. Louis, MO). Bisindolylmaleimide I was purchasedfrom EMD Biosciences (La Jolla, CA). On receipt, allreagents were dissolved in dimethyl sulfoxide (DMSO)as stock solutions. When these agents were used in anexperiment, equal volumes of DMSO were added to aparallel group as control.

Cell Line

Human prostate cancer ARCaPE cells [15,16] werecultured in T-medium (Invitrogen, Carlsbad, CA)containing 5% fetal bovine serum (FBS, Atlanta Bio-logicals, Lawrenceville, GA), penicillin (100 U/ml),and streptomycin (100 mg/ml). Cells were cultured ina humidified incubator at 378C with atmospheric O2

supplemented with 5% CO2.

Assay for Cell Proliferation

The protocol for assaying cell proliferation wasreported previously [8]. In this study, cells were platedonto 96-well plate at a density of 2.5� 104/100 mlmedium in each well for 24 hr. The cells were thentreated in triplicate with different concentrations ofPMA for 48 hr before subjected to proliferation assay.

Assays for CellMigration

Two methods were used to assay for cell migration.The protocol for assaying cell migration using theBoyden chamber assay was reported previously [8]. Toevaluate cell migration by the scratch wound healingmethod, cells on a six-well plate were allowed to growto full confluence. A scratch wound was made bydenuding a streak of the cell monolayer with a sterilepipette tip 2.6 mm in diameter. The culture was treatedwith 100 nM PMA for 96 hr. Migration of the cellsinto the denuded space was documented by micro-photography.

Assay for Cell Invasion

The assay method for cell invasion was reportedpreviously [12]. In this study, cells were treated with

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100 nM PMA for 48 hr in the Transwell inserts coveredwith 100 ml Matrigel (BD Biosciences, San Jose, CA).Cells that invaded to chamber and on the outer surfaceof the insert were collected and subjected to MTT assay.

Western Blotting

The Western blotting protocol used was reportedpreviously [8]. In this study, antibodies to E-cad,vimentin, cytokeratin 18 (CK-18), b-actin, and allthe secondary antibodies conjugated with horserad-ish-peroxidase were purchased (Santa Cruz Biotech-nology, Santa Cruz, CA). Specific signals were detectedwith the ECL plus Western blotting detection kit (GEHealthcare Bio-Sciences, Piscataway, NJ).

Microphotography

Cell images were documented with a Nikon E300inverted microscope equipped with a MagnaFiredigital camera.

RESULTS

The phorbol ester PMA is a specific PKC activator[17] and a potent tumor promoter in experimental mice[18–20]. At the cellular level, PMA promotes migrationand invasion in established cell lines [21,22]. Becauseincreased migration and invasion in ARCaPE cells areoften accompanied by EMT, we assessed whether PMAcould induce EMT in these cells.

TreatmentWithPMAResultsinEMTinARCaPECells

EMT-associated morphologic changes in ARCaPE

cells have been described in detail [6,8,12]. Whencultured in vitro, ARCaPE cells display distinctepithelial morphology, in pentagonal shapes with tightintercellular junctions, forming a cobblestone-likeclonal organization. After committing to EMT, ARCaPE

cells adopt the morphology of mesenchymal stromalcells, becoming dissociated from each other, losingcellular polarity, and adopting spindle-like shapes. Themorphologic change is followed by increased cellularmotility, as ARCaPE cells committed to EMT displayedmigratory and invasive behavior.

To assess whether PMA induces EMT, we treatedARCaPE cells with different concentrations of PMA,and inspected cellular changes daily for signs of EMT-like morphologic changes. These experiments revealedthat the phorbol ester could effectively induce EMT-like changes in ARCaPE cells. Whereas PMA at 1 nMdid not cause morphologic changes, ARCaPE cellstreated with 10 nM PMA became enlarged and flat(Fig. 1). Importantly, PMA at 50 and 100 nM inducedcomplete morphologic changes in 24 hr (Fig. 1). Com-pared to a control group, the treated cells separated

from each other, became spindle shaped, and lostcellular polarity. These cells rearranged in all directionsand stacked on each other when cell density increasedalong with the treatment time. The results from thesestudies indicated that PMA-induced EMT-like changesin ARCaPE cells.

From four repeated experiments, we found twointeresting features of the PMA-induced EMT-likechanges. First, the effect of PMA is time-dependent.In every experiment performed, no morphologicalchanges were observed in ARCaPE cells in the first8 hr of treatment. The morphological changes appearedgradually afterwards, with cell shape changes becom-ing discernible at 16 hr, and all the cells exhibitedmesenchymal stromal morphology at 24 hr (Fig. 1).Thereafter, the morphologic change was sustained aslong as PMA was present. Second, although the effect ofPMA seemed dose dependent within the 100 nM range(Fig. 1), higher concentrations of PMA (200, 500,and 1,000 nM) did not cause additional morphologicchanges, nor did these concentrations accelerate therate of morphologic change. This observation indicatedthat the dose effect of PMA was limited by intracellularfactors, probably by the availability of PKC proteins.

The EMT in ARCaPE cells could be irreversible, sinceARCaPE clones retrieved from xenograft tumors dis-played permanent EMT features [14]. To determinewhether the PMA-induced changes were reversible, werecovered the ARCaPE cells that were treated with100 nM PMA for 96 hr. After being detached by trypsinand washed in PBS, the cells were cultured in theabsence of PMA. Displaying spindle-shaped stromalmorphology at beginning of the culture, these cellsunderwent cell division to form colonies that werecompletely epithelial in cell shape and colony organ-ization, indistinguishable from untreated ARCaPE

cells. PMA-induced EMT in ARCaPE cells is thusreversible. In experiments to assess the effect on cellgrowth and survival, PMA did not induce significantchanges in cell growth at doses from 50 to 1,000 nM. Onthe other hand, PMA in this dose range did not causesignificant cell death within 96 hr of the treatment. Itseems that EMT is a specific response of the ARCaPE

human prostate cancer cells to the PMA treatment.

PMAPromoted EMTinARCaPECells byActivating PKCIsoenzymes

The PKC family comprises three subfamilies ofisoenzymes [17,23,24], while PMA is a known activatorof both the conventional (a, bI, bII, and g isoenzymes)and novel (d, e, h, and y isoenzymes) PKC subfamiliesin the 1–100 nM range. To confirm that PMA promotedEMT in ARCaPE cells by activating PKC, we usedspecific PKC inhibitors to block the effect of PMA. A

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PMAActivates PKCto Induce EMT 1121

general PKC inhibitor, Go6983, was used at 200 nM inorder to inhibit all the PKC isoenzymes. This inhibitionled to significant inhibition of EMT in ARCaPE cells(Fig. 2). A well-characterized isoenzyme-specific inhib-itor, bisindolylmaleimide I, was known to inhibitconventional PKC isoenzymes at 20 nM, and to inhibitnovel PKC isoenzymes at much higher concentrations[25,26]. Bisindolylmaleimide I at 50 nM did not showany inhibitory effect, whereas at 1,000 nM it completelyprevented PMA-induced EMT in ARCaPE cells (Fig. 2).These results supported that PMA promoted EMT inARCaPE cells by activating PKC, and certain isoen-zymes of the novel PKC subfamily may be mediatingthe promoting effect. In addition, the PKC inhibitorstested in this study did not cause death in ARCaPE cellsafter a 96 hr treatment, suggesting that PKC isoen-zymes are not involved in maintaining the survival ofARCaPE cells.

PMAInhibits Expression of EpithelialMarkersbutInducesMesenchymal StromalMarkers

Besides morphologic changes, EMT in ARCaPE

cells is accompanied by reduced expression of

epithelial markers and by activated expression ofstromal markers [6,12]. Accordingly, we examinedthe effect of PMA on EMT-related marker proteinexpression (Fig. 3). ARCaPE cells treated with 100 nMPMA for different times were sampled for Westernblotting with specific antibodies to E-cad, CK-18, andvimentin. These analyses revealed that PMA inhibitedthe expression of E-cad, a critical surface proteinmaintaining intercellular junction between epithelialcells. The inhibition was a time-dependent process.Though there was no change in the level of E-cadwithin the first 8 hr, the level of E-cad decreasedafterwards as treatment prolonged, and almost com-pletely disappeared after 72 hr of PMA treatment.The decreased E-cad expression was accompaniedby reduced expression of CK-18, an epithelial cell-specific intermediate filament protein. Concurrently,expression of the stromal cell-specific intermediatefilament protein vimentin was activated (Fig. 3). Theseresults indicated that PMA treatment suppressedexpression of epithelial cell marker proteins andstimulated expressional activity of the stromalmarker.

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Fig. 1. PMAisapotentinducerofEMT.Intheseexperiments,humanprostatecancerARCaPEcellsweretreatedwithdifferentconcentrationsof PMAfor 24hr.ARCaPEcells inregularculture (PMA,0nM)displayepithelialmorphology,withpentagonal cells in tightintercellular junctionforming cobblestone-like cellular organization.PMA in the100nMrange induceddose-dependentmorphologic changes.Whereas lower con-centrations (PMA,1nM and PMA,10nM) inducedpartialmorphologic changes, a higher dose (PMA,100nM) induced complete conversion ofARCaPE cells to mesenchymal stromal morphology, suggesting EMT. PMA at 500 and 1,000nM even higher concentrations (PMA, 500 and1,000nM)didnotcauseadditionalmorphologicchanges.Theresultisrepresentativeof fourrepeatedexperiments.Phasecontrastmicrophoto-graphsare shownat100�magnification.

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PMAPromotesMigration and Invasion ofARCaPECells

PMA is well known for its function of promoting cellmotility and scattering [21,22], and EMT in ARCaPE

cells was accompanied by increased migration andinvasion [12]. We assessed the effect of PMA on themotility of ARCaPE cells. Two methods, scratch woundhealing and the Boyden chamber assay, were used todetect changes in cell migration. In both assays, PMAshowed a marked simulating effect on ARCaPE cellmigration (Fig. 4A,B). A similar stimulating effect wasobserved in the cell invasion assay, in which PMAinduced significantly accelerated invasion of ARCaPE

cells through the Matrigel (Fig. 4C). In these assays, thePMA-induced cellular motility could be inhibitedeffectively by the PKC inhibitor bisindolylmaleimide Iat 1,000 nM (Fig. 4B,C). Bisindolylmaleimide I at 50 nMdid not show any inhibition. These results suggestedthat the increased motility of ARCaPE cells wasmediated by activation of the novel PKC isoenzymes.

PMA-Induced EMTinARCaPECells Is DependentonGeneTranscription and Translation

We noticed that in both the process of morphologicalchanges and the process of expression alterations,PMA-induced EMT had a latent phase of more than8 hr. The delay in EMT could be caused by the newtranscription and translation of additional genesrequired to execute the EMT process. To explore therole of new gene transcription and translation in EMT,we added Act.D, a specific inhibitor of gene tran-scription, together with PMA to ARCaPE cells. Com-pared to the control group, in which PMA inducedmarked EMT at 48 hr, cells with the combinatorytreatment failed to adopt mesenchymal stromal cellmorphologies (Fig. 5). Similar results were obtainedwhen cyclohexamide (CHX), a specific inhibitor ofprotein translation, was used in combination with PMA(Fig. 5). As inhibitors for transcription and translation,both Act.D and CHX showed cytotoxicity to ARCaPE

cells after 24 hr, and beyond 48 hr many cells started todie. Nonetheless, none of the remained cells adaptedstromal morphology even after 96 hr of treatment.These results suggested that that PMA-induced EMT

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Fig. 2. PMAinducesEMTbyactivatingPKC.ARCaPEcellswere treatedwithPMA(100nM)inthepresenceofPKCinhibitorGo« 6983orBisin-dolylmaleimide I for 48hr.Go« 6983 at 200nM inhibited EMT. A lowconcentration of Bisindolylmaleimide I (50nM) did not show an inhibitoryeffect, whereas Bisindolylmaleimide I at 1,000nM completely prevented EMT, indicating that members of the novel PKC subfamily weremain mediators in PMA-induced EMT.The result is representative of three repeated experiments. Microphotographs are shown at 100�magnification.

Fig. 3. PMAinhibits epithelialmarkerE-cadexpression andindu-ces stromal marker vimentin. ARCaPE cells treated with PMA(100nM) were sampled at different times (hr.) for Western blotanalysis of EMT markers. The result is representative of tworepeatedexperiments.

PMAActivates PKCto Induce EMT 1123

requires transcriptional activation and EMT functionsrequire newly synthesized proteins.

DISCUSSION

By applying PMA treatment to the well-establishedEMT model of human prostate cancer ARCaPE cells, wefound that PMA is a potent promoter for EMT-likephenotypes. PMA-induced ARCaPE cells to undergomarked morphologic change from epithelial to mesen-chymal stromal cell shapes (Fig. 1). The morphologicchanges were accompanied by expressional changes, inwhich ARCaPE cells lost the epithelial markers E-cadand CK-18, and switched on the expression of stromalintermediate filament protein vimentin (Fig. 3). Loss ofE-cad and the activated vimentin expression are thetwo most informative markers of EMT [27]. In addition,

PMA-treated ARCaPE cells showed significantlyincreased motility, both in assays for cell migrationand cell invasion (Fig. 4), supporting the conclusionthat PMA is a potent inducer of EMT in ARCaPE

prostate cancer cells. We observed similar morphologicchanges in MCF-7 breast cancer cells upon PMAtreatment (data not shown). Although PMA is knownto be a stimulator of cell motility and scattering, fewstudies have reported accompanying morphologicchanges caused by PMA. This work is the first todemonstrate that PMA is an EMT inducer.

PMA, when applied in vivo, is a potent promoterfor mouse skin tumorigenesis [18–20]. The effectof PMA is mainly through mimicking diacylglycerol,a lipid metabolite of the cytoplasmic membrane andnatural second messenger activating PKC isoenzymes.As a potent agonist, PMA triggers autophosphoryla-

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Fig. 4. PMApromotes cellmigration andinvasion.Cellmigrationwas assayedby twomethods.A: in a scratchwoundhealing assay,ARCaPE

cells treatedwith100nMPMAwere recorded for cellmigrationbymicrophotography.The result is representative of three repeated experi-ments.Microphotographs are shown at 40�magnification.B: in a BoydenChambermigration assay,ARCaPE cells grownon the insertsweretreatedfor96hrwith100nMPMA(PMA),1,000nMBisindolylmaleimideI(Bis.I),oracombinationof thetwo(PMAþBis.I).Cellsmigratingacrossthe filterswere collectedandquantifiedbyMTT conversion.Eachbar represents themeanof triplicate assays from threeBoydenChambers.The result is representative of two separate experiments.C: to determine cell invasion, ARCaPE cells grown on Matrigel-coated insertswere subjected to the same treatment.Cellsmigrating through theMatrigel-coated filterswere collected andquantifiedbyMTTconversion.Eachbarrepresents themeanof triplicateassays fromthreeBoydenChambers.Theresultisrepresentativeof two separateexperiments.

1124 He et al.

tion of both conventional and novel PKC isoenzymes.The activated PKC in turn functions as a serine andthreonine kinase to catalyze phosphorylation of a widepanel of regulatory proteins [24]. Using a generalPKC inhibitor and an isoenzyme-specific inhibitor, wedemonstrated that PMA-induced EMT in ARCaPE cellsby activating novel PKC isoenzymes (Fig. 2). Addi-tional studies are needed to identify which of the novelPKC isoenzymes is the principal mediator for EMT.Further investigation is warranted to elucidate themechanism of the isoenzymes in mediating EMT.

The expression level and catalytic activity of PKCisoenzymes are correlated to cancer progression andtumor metastasis [23]. Members of the novel PKCsubfamily have been known to promote cell migrationand invasion in other cell lines [28–30]. Strategiestargeting specific PKC isoenzymes are shown to reducetumor growth and metastasis [28,31–35]. It would beintriguing to investigate whether specific targetingstrategies could be developed to target-specific iso-enzymes to prevent tumor cells from committing toEMT. On the other hand, we have reported that EMT inARCaPE cells is controlled by transcription factorsincluding SNAIL, a substrate of serine and threoninekinase [12]. It is intriguing to investigate whether PMA-activated novel PKC isoenzymes causes phosphoryla-tion and nuclear localization of these transcriptionfactors.

We found that PMA effectively suppressed E-cadprotein expression in a time-dependent manner (Fig. 3).Further studies have to be conducted to determine themechanism by which PMA suppresses E-cad expres-

sion. As a cytoplasmic membrane protein, E-cad has adual function [36–38]. Besides functioning as a struc-tural protein for intercellular junctions, E-cad seques-ters b-catenin to prevent it from translocation tonucleus, where b-catenin functions as a transcriptionalco-regulator to activate a panel of stromal-specificgenes. In ARCaPE cells, it is possible that afteradministration of PMA and upon loss of E-cadexpression, b-catenin undergoes nuclear translocationto activate the expression of stromal-specific genes,which function to cause the dramatic morphologic andbehavioral changes observed in ARCaPE cells. Thisstudy demonstrated that inhibiting either new genetranscription or new protein synthesis could inhibitEMT (Fig. 5), strongly indicating that the stromalphenotype was supported by newly expressed stro-mal-specific genes. PMA-treated ARCaPE cells shouldbe an ideal model for studying the expression ofstromal-specific genes during EMT.

ACKNOWLEDGMENTS

This work is supported by research grantsR21CA112330, PC040578, CA132388 (R.X.W.), andCA98912-02 (L.W.K.C.).

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Fig. 5. PMA-induced EMTrequires gene transcription and newly synthesized proteins. ARCaPE cells were treated for 48hr with PMA(100nM) in thepresenceofAct.D,aninhibitorofgene transcription,orCHX,aninhibitorofprotein synthesis.Comparedto thecontrolgroup,bothAct.D andCHXinhibitedARCaPE cells fromadoptingmesenchymal stromalmorphology.Theresult is representative of threerepeatedexperiments.Microphotographsare shownat100�magnification.

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