The inhibition of sphingomyelin synthase 1 activity induces collecting duct cells to lose their epithelial phenotype

Accepted Manuscript The inhibition of sphingomyelin synthase 1 activity induces collecting duct cells to lose their epithelial phenotype

Yamila Romina Brandán, Edith del Valle Guaytima, Nicolás Octavio Favale, Lucila Gisele Pescio, Norma B. Sterin-Speziale, María Gabriela Márquez PII: DOI: Reference:

S0167-4889(17)30294-X doi:10.1016/j.bbamcr.2017.11.004 BBAMCR 18207

To appear in: Received date: Revised date: Accepted date:

16 May 2017 30 October 2017 7 November 2017

Please cite this article as: Yamila Romina Brandán, Edith del Valle Guaytima, Nicolás Octavio Favale, Lucila Gisele Pescio, Norma B. Sterin-Speziale, María Gabriela Márquez , The inhibition of sphingomyelin synthase 1 activity induces collecting duct cells to lose their epithelial phenotype. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbamcr(2017), doi:10.1016/ j.bbamcr.2017.11.004

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ACCEPTED MANUSCRIPT The inhibition of sphingomyelin synthase 1 activity induces collecting duct cells to lose their epithelial phenotype 1

Yamila Romina Brandán , Edith del Valle Guaytima 1, Nicolás Octavio Favale 2,3, Lucila Gisele Pescio 2,3, Norma B. Sterin-Speziale 2, and María Gabriela Márquez 1* 1

Instituto de Investigaciones en Ciencias de la Salud Humana (IICSHUM), Universidad

Instituto de Química y Físico-Química Biológica (IQUIFIB) -CONICET, Facultad de

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Nacional de La Rioja, Av. Luis Vernet 1000, (5300) La Rioja, ARGENTINA

Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, (C1113AAD) Buenos

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Aires, ARGENTINA

Cátedra de Biología Celular, Departamento de Ciencias Biológicas, Facultad de Farmacia

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y Bioquímica, Universidad de Buenos Aires, Junín 956, (C1113AAD) Buenos Aires,

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ARGENTINA

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E-mail address

Yamila Romina Brandán: [email protected] Edith del Valle Guaytima: [email protected]

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Nicolás Octavio Favale: [email protected] Lucila Gisele Pescio: [email protected]

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Norma B. Sterin-Speziale: [email protected]

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María Gabriela Márquez: [email protected]

To whom correspondence should be addressed: Dr. María Gabriela Márquez, Instituto de Investigaciones en Ciencias de la Salud Humana (IICSHUM), Universidad Nacional de La Rioja, Av. Luis Vernet 1000, (5300) La Rioja, ARGENTINA, Phone: 054 03804438683, Email: [email protected]. Running head: Sphingomyelin synthesis is essential for cell adhesion structures

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ACCEPTED MANUSCRIPT Abstract Epithelial tissue requires that cells attach to each other and to the extracellular matrix by the assembly of adherens junctions (AJ) and focal adhesions (FA) respectively. We have previously shown that, in renal papillary collecting duct (CD) cells, both AJ and FA are located in sphingomyelin (SM)-enriched plasma membrane microdomains. In the present

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work, we investigated the involvement of SM metabolism in the preservation of the

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epithelial cell phenotype and tissue organization. To this end, primary cultures of renal papillary CD cells were performed. Cultured cells preserved the fully differentiated

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epithelial phenotype as reflected by the presence of primary cilia. Cells were then incubated for 24 h with increasing concentrations of D609, a SM synthase (SMS) inhibitor. Knock-

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down experiments silencing SMS 1 and 2 were also performed. By combining biochemical and immunofluorescence studies, we found experimental evidences suggesting that, in CD

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cells, SMS 1 activity is essential for the preservation of cell-cell adhesion structures and therefore for the maintenance of CD tissue/tubular organization. The inhibition of SMS 1

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activity induced CD cells to lose their epithelial phenotype and to undergo an epithelial-

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mesenchymal transition (EMT) process.

Keywords: collecting duct cells, sphingomyelin synthase, epithelial-mesenchymal

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transition, adherens junctions

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ACCEPTED MANUSCRIPT 1. Introduction In the renal papilla, the collecting ducts (CD), which are comprised of epithelial cells, are surrounded by interstitial tissue, which includes an extracellular matrix (ECM) in which fibroblasts are present. In mammals, the ability of epithelial cells to join to each other and to the ECM is important in a high number of biological processes, including

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embryogenesis, wound healing, and maintenance of tissue integrity and function. In

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epithelial tissue, cell-cell adhesions are mediated by adherens junctions (AJ) by proteins called E-cadherins, which span the plasma membrane and contact E-cadherins of other

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cells. At sub-membrane sites, β-catenin is anchored to the cytoplasmic tail of E-cadherin and, via α-catenin and vinculin, connects with the actin cytoskeleton inside the cell [1, 2, 3,

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4, 5, 6]. The attachment of cells to the ECM is mediated by multiprotein structures socalled focal adhesions (FA) [7, 8]. FA assembly occurs through the binding of the integrin-

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extracellular domain to ECM proteins, followed by the interaction of the β-integrin cytoplasmic domain with talin. Talin can then recruit vinculin, which, in turn, binds other

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FA proteins [7, 8]. Both vinculin and talin bind to F-actin thus connecting the actin cytoskeleton to ECM components [7]. FA are dynamic structures that assemble and

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disassemble depending on the needs of the cell [9, 10]. During the first stage of the tissue organization process, FA formation precedes cell-cell junction since, during the initiation of

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AJ formation, cells must migrate to contact neighboring cells [11]. Vinculin is expressed in most cell types [12] and, as mentioned above, it works as a

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scaffold protein that links FA and AJ proteins with the actin cytoskeleton, thus regulating FA and AJ dynamics [13]. It is accepted that vinculin exists in a cytosolic and in a

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cytoskeletal pool, considering as the cytosolic pool the one formed by an auto-inhibited soluble conformation of the protein [14, 15, 16]. Studies from our laboratory have demonstrated the existence of an additional vesicle-associated pool, which in turn serves to reconstitute vinculin to FA [17, 18]. Sphingomyelin (SM) is the main sphingolipid in mammalian cells. Besides providing a structural framework for plasma membrane organization, sphingolipids regulate various cellular processes such as growth, death, differentiation, and intracellular trafficking [19]. SM is biochemically synthesized through the activity of serine-palmitoyl-CoA transferase, 3-ketosphinganine reductase, ceramide synthase, dihydroceramide desaturase, and SM 3

ACCEPTED MANUSCRIPT synthase (SMS). SMS, which uses ceramide and phosphatidylcholine as substrates, is the last enzyme in SM biosynthesis [20]. Two isoforms, SMS 1 and SMS 2, have been cloned in mammals [21]. SMS 1 localizes in the Golgi apparatus, whereas SMS 2 can be localized in the plasma membrane but also in the Golgi apparatus [22]. SM synthesis takes place on the luminal side of the trans Golgi network, by SMS 1, and in the extracellular leaflet of the plasma membrane, by SMS 2 [21, 23, 24]. Due to the localization and activity of the

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enzymes, the membranes of the trans Golgi network, the plasma membrane and even the

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endosomal recycling compartment are enriched in SM [25]. In recent years, genome studies

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have revealed a number of genes involved in lipid membrane dynamics linked to several neurological pathologies [26, 27, 28]. In previous works, we have demonstrated that, in CD cells, FA and AJ protein complexes are located in SM-cholesterol-rich detergent-resistant

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microdomains or rafts, whose lipid composition is necessary to preserve cellular adhesion

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structures [29, 30].

In most animals, the mesenchymal–epithelial transition (MET) regulates early stages of

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development, and a number of epithelial sheets, including the tubular cells of the kidney originate from mesenchymal cells [31, 32]. On the other hand, changes in cell phenotype

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between epithelial and mesenchymal states, defined as epithelial–mesenchymal transition (EMT), have an important role during development [33, 34]. The reactivation of EMT in

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the adult is regarded as a physiological attempt to control inflammation, wound healing and tissue regeneration [35, 36]. However, EMT is also involved in the generation of fibrosis

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and the progression and metastasis of cancers [33, 34, 35, 36]. Taking into account that, in renal papillary CD cells, both AJ and FA are located in SM-

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enriched plasma membrane microdomains, in the present work, we investigated the possible implication of SM metabolism in cellular fate. By combining biochemical and immunofluorescence studies, we found experimental evidence suggesting that, in CD cells, SMS 1 activity is essential for the preservation of cell-cell adhesion structures and therefore for the maintenance of CD tissue/tubular organization.

2. Materials and methods

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ACCEPTED MANUSCRIPT 2.1 Antibodies and reagents- Monoclonal vinculin, β-catenin, acetylated tubulin and polyclonal α-catenin antibodies were purchased from Sigma-Aldrich. Polyclonal Ecadherin and monoclonal vimentin antibodies were from Santa Cruz Biotechnology Inc. Monoclonal antibodies against α-smooth muscle actin (α-SMA) and cytokeratin 7 (CK7) were from Leica Biosystems. Goat anti-rabbit and goat anti-mouse secondary antibodies conjugated to either TRITC or FITC were from Jackson ImmunoResearch Inc. The avidin-

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biotin-peroxidase kit was from Vector Laboratories and 3,3´diaminobenzidine from Sigma-

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Aldrich. The SMS activity inhibitor tricyclodecan-9-yl-xanthogenate (D609) was from

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Sigma-Aldrich. The N-lauroyl-D-erythro-sphingosylphosphorylcholine (C12-SM) was from Avanti Polar Lipids, Inc. All culture reagents were from Gibco, Invitrogen. All other

purchased from local commercial suppliers.

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reagents and chemicals, unless otherwise stated, were from Sigma-Aldrich or Merck, and

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2.2 Animals and tissue preparation- Male Wistar rats (250-300g) were housed in a lightcontrolled room with a 12:12 h light-dark cycle and allowed free access to water and

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standard rat chow. All animals were handled according to the rules for animal care and use of laboratory animals of the National University of La Rioja, Argentina. The animal

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protocol was reviewed and approved by the Ethics Committee for the Care and Use of Laboratory animals, National University of La Rioja (CICUAL-UNLaR). Animals were

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killed by cervical dislocation, kidneys removed and renal papillae isolated and collected on ice-cold 10 mM Tris-HCl, pH 7.4, containing 140 mM NaCl, 5 mM KCl, 2 mM MgSO4, 1

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mM CaCl2 and 5.5 mM glucose (TBS). 2.3 Cell cultures and treatments- Primary cultures of papillary CD cells were performed

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according to Stokes et al. [37]. Briefly, renal papillae were minced to 1-2 mm3 pieces and incubated at 37°C in sterile TBS containing 0.1 % collagenase II under 95%O2 / 5%CO2. After 70 min, digestion was stopped and isolated cells and structures were separated by centrifugation at 175g for 10 min. The crude pellet containing most papillary cell types, tubular structures and tissue debris was washed twice and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) with F-12 (1:1), 10 % fetal bovine serum (Natocor), 100 U/ml penicillin and 100 µg/ml streptomycin. The enriched CD pellets were obtained by centrifugation at 60g for 1 min and resuspended in an adequate volume of DMEM/F12. Enriched-tubular suspensions were seeded in sterile dry-glass coverslips placed in six-well 5

ACCEPTED MANUSCRIPT multidishes or directly on the multidishes for scraping experiments. In the experiments performed in the presence of the SMS inhibitor, after 72 h of culture, the cells were treated with D609 at a concentration of 20, 50 and 100 µM for additional 24 h. It is important to note that these D609 concentrations were adjusted to inhibit SMS activity and maintain cell viability. In rescue experimentes, the cells were treated with D609 at a concentration of 50 μM in the presence of exogenous 10 μM C12-SM for 24 h. Incubations were stopped on ice

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and cells rapidly processed for microscopy or scrapping experiments.

2.4 Cell surface biotinylation- Cells were cultured as described above. After 72 h of

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culture, cells were treated with D609 at a concentration of 50 µM for 24 h. After washing with PBS containing 0.1 mM CaCl2 and 1 mM MgCl2, cells were incubated on ice twice

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with NHS-SS-biotin (Pierce, Life Technologies, TM) (0.5 mg/ml in PBS/0.1 mM CaCl2 - 1 mM MgCl2) for 20 min, quenched for 15 min with PBS containing 50 mM NH4Cl, and

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extensively washed with PBS/0.1 mM CaCl2 -1 mM MgCl2. Biotinylated cells were lysed for 30 min in lysis buffer (150 mM NaCl, 20 mM Tris pH 8, 1 mM EDTA, 1% Triton X-

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100, and protease inhibitor cocktail), and passed through a 28-gauge needle. Aliquots of the material were used as total homogenates. The cell lysates were centrifuged at 14,000g for

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20 min and the protein content determined by the method of Lowry. Aliquots of the supernatants (1 mg proteins) were incubated overnight at 4 °C with 45 µl of a streptavidin-

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agarose bead suspension to obtain the bound fractions. The bound fractions were washed first with washing buffer (150 mM NaCl, 20 mM Tris pH 8, 1 mM EDTA) containing 1%

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Triton X-100, and later with the same buffer without Triton. The bound material was eluted from the beads with 40 µl of Laemmli sample buffer. Aliquots of the total homogenate and

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the unbound fraction (100 µg proteins) were precipitated with cold acetone at 80 % as described in section 2.5. The bound and total homogenate fractions were resolved in an 8 % SDS-Polyacrylamide gel under reducing conditions, and transferred to polyvinylidene difluoride (PVDF) membranes [38, 39]. 2.5 Protein precipitation- Cells were cultured as described above. After washing with PBS, cells were resuspended in a solution of 0.25 M sucrose containing 25 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 2 mM EGTA, and 1 mM PMSF. Then, cells were scrapped off and passed through a 28-gauge needle. The resulting homogenate was centrifuged at 860g for 10 min. Aliquots of the postnuclear supernatants were used as starting materials and the 6

ACCEPTED MANUSCRIPT protein content was determined by the method of Lowry. Acetone precipitation was performed according to Crowell et al. [40]. Briefly, NaCl was dissolved in the samples (50 µg proteins), reaching a concentration of 10 mM. Then, a volume of cold acetone at 80 % was added. Samples were incubated overnight at -20°C, and then centrifuged at 16,000g for 20 min. The supernatant was carefully removed, leaving behind less than 20 µl of solution. An additional washing step was performed by adding the corresponding volume of cold

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acetone at 80 % to the pellet, and removing the bulk of supernatant following centrifugation

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(16,000g for 20 min). The residual acetone was removed from the pellets by air dying.

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2.6 Immunoblot analysis– The pellets obtained by acetone precipitation were resuspended in PBS and incubated with 4X Laemmli buffer at 100°C for 5 min, and resolved in an 8 %

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SDS-Polyacrylamide gel under reducing conditions, and transferred to PVDF membranes. After blotting, the membranes were treated with 5% non-fat milk in TBS-Tween 20 and

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incubated with the indicated antibodies. Primary interaction was evidenced by using the avidin-biotin-peroxidase and 3,3´diaminobenzidine reaction. To control the protein loading

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of samples, membranes were stained with Coomassie blue. The blots were scanned and

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signals quantified by optical densitometry with Gel-Pro Analyzer 3.1. 2.7 Cell transfection- Cell cultures were transfected with specific siRNA for the two SMS isoforms. To obtain SMS siRNA-transfected cells, primary cultures of papillary CD cells

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were seeded and transfected at 48 h of the culture either with 40 nM of SMS 1 siRNA duplex

5´-ACCUGUUGCACCGAUAUUCAATT-´3

5´-

lupus

familiaris

duplex

5´-

GAGTCTCCGTTGAGCTTTGG-3 5´-GAGTCTCCGTTGAGCTTTGG-3´ (Gene

ID:

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Canis

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UUGAAUAUCGGUGCAACAGGUTT-´3 (Gene ID: 477582, sphingomyelin synthase 1, )

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with

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nM

SMS

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siRNA

478505, sphingomyelin synthase 2, Canis lupus familiaris) (Invitrogen) by using HiPerFect Transfection Reagent (QUIAGEN), following the manufacturer´s protocol. Efficacy was determined by cotransfection with a nonsilencing siRNA conjugated with AlexaFluor 488 (AllStars Negative Controls-QUIAGEN). This siRNA was also used as a control and to monitor the transfection. Positive intracellular green dots were considered as transfected. The transfection efficacy was between 15% and 25%. Cells were fixed with 2% paraformaldehyde as described and incubated with an antibody against α-catenin. Labeled

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ACCEPTED MANUSCRIPT cells were analyzed by immunocytochemistry for the evaluation of morphological changes induced by specific siRNAs. 2.8 Cell labeling and immunofluorescent microscopy- For immunostaining, cells treated as described above were washed with PBS and fixed either with methanol (at -20°C for 10 min) and acetone (at -20°C for 4 min) or with 2% (w/v) paraformaldehyde at 25°C for 20

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min, and permeabilized with 0.1 % (v/v) Triton X-100 at 25°C for 20 min. After fixation,

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cells were washed with PBS and incubated with 3% bovine serum albumin (BSA, Calbiochem) in PBS at 25°C for 60 min. Then, cells were incubated with the appropriate

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combinations of antibodies overnight at 4ºC in 3% BSA in PBS. Mouse and rabbit primary antibodies were detected using fluorescent FITC- or TRITC-conjugated goat anti-mouse or

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anti-rabbit antibodies. Nuclei were counterstained with Hoechst 33258. Finally, the cells were mounted using Vectashield Mounting Media (Vector Laboratories) and stored at 4°C

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until analysis.

2.9 Image analysis- Specimens were examined with an Olympus FV300 Confocal

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Microscope (Model BX61), with the acquisition software FluoView version 3.3 provided

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by the manufacturer. Cells were examined with a 60X Apo oil objective (1.40 NA). Double fluorescence for green and red channels was visualized by using an argon-helium-neon laser. Double-stained confocal images were obtained by sequential scanning for each

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channel to eliminate the crosstalk of chromophores and ensure the reliable quantification of colocalization. Confocal images represent a single Z section. All images were obtained with

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a cooled CCD camera (and processed for output purposes using Adobe Photoshop

USA).

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software). Images were analyzed using Image-Pro plus version 5.1.2 (Media Cybernetics,

In other experiments, cells were examined with a conventional fluorescence Olympus IX81 microscope with 40X Uplan FLN (0.75 NA air) objective. Images were acquired with an Olympus DP70 Digital Camera, with the acquisition software provided by the manufacturer. For siRNA analysis, cells were also observed with a phase contrast objective. All images were processed for output purposes by using Adobe Photoshop software. When necessary, we applied filters to soften the images with Image-Pro Plus Version 5.1.2 (Media Cybernetics, USA). 8

ACCEPTED MANUSCRIPT All FA in 5-10 randomly selected cells were analyzed to generate quantitative data sets for each treatment. After spatial calibration, the pixel length resulted in 0.3125 µm. To reduce background variations in our immunofluorescence images, we applied a “flatten filter”. This is often done to prepare an image for count/size operations because flattening reduces the intensity variations in the background pixels. To select the intensity range of the objects (FA) to be counted, we applied the software command to perform the “segmentation” of the

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image. This application segments the image into objects and background. The total number

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of vinculin- and talin-immunostained FA per cell in internal and peripheral cells were

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calculated. To this end, the contour of each cell in the image (area of interest) was manually drawn by using the mouse pointer of the program Image-Pro Plus Version 5.1.2.

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To evaluate the colocolaization between AJ proteins, “segmentation” process was performed using the Image-Pro Plus Version 5.1.2 module. In this process, the

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colocalization areas appear in white over a black background. 2.10 Quantitative colocalization analysis. The confocal images were analyzed using the

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Image-Pro Plus version 5.1.2. All cell-cell adhesion sites (adherens junctions) in 6-8 randomly selected cells were analyzed for generation of quantitative data sets for the 20, 50

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and 100 μM D609 treatment, and three independent experiments were performed. Colocalization between vinculin and α-catenin was evaluated quantitatively by the

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colocalization command of the Image-Pro Plus version 5.1.2. The Manders Overlap Coefficient (MOC) was used to estimate the degree of colocalization. Its value is defined

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from 0 to 1, and a MOC ≥ 0.7 indicates colocalization and implies that 70% of both its components overlap with the other part of the image. Three independent experiments were

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performed.

2.11 Quantitative analysis of vimentin- and α-SMA-immunostained CD cells- Cells were examined with a conventional fluorescence Olympus IX81 microscope with 40X Uplan FLN (0.75 NA air) objective. Total and vimentin- or α-SMA-immunostained cultured CD cells were recordered to generate quantitative data sets for each treatment. Results are expressed as percentage (mean ± SEM), and three independent experiments were performed. The number of vimentin- or α-SMA-immunostained overlapping cells was also recordered in randomly selected microscopic fields. Results are expressed as number of positive cells per field (mean ± SEM), and three independent experiments were performed. 9

ACCEPTED MANUSCRIPT 2.12 Statistical analysis - The statistical analysis was performed using GraphPad Instat version 3.01 (GraphPad Software, Inc). Results are expressed as the mean ± SEM. Data from control and different treatments were analyzed by unpaired t-test and unpaired t-test with Welch correction when standard deviations were considered not equal. Statistical

3. Results

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3.1 The inhibition of SMS 1 impairs cell-cell adhesion

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significance was set at p<0.05.

Taking advantage of the fact that CD primary cultured cells retain many characteristics of

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their behavior in intact tissue, primary cultures of renal papillary CD cells were performed. The phase contrast images show the typical morphology of cultured CD cells displaying a

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well spread morphology (Fig. 1A). When cultured, these cells preserve the fully differentiated epithelial phenotype as reflected by the presence of a primary cilium [41],

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which can be visualized with an anti-acetylated tubulin antibody (Fig. 1B-B', arrows) and

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the expression of the epithelial cell marker CK7 (Fig. 1C) At 72 h of culture, when cells were joined together, cells were incubated for additional 24 h

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with increasing concentrations of the SMS inhibitor D609 (20, 50 and 100 μM), and cell– cell adhesion structures were studied. Since α-catenin links the cadherin-catenin complex

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directly to the actin cytoskeleton, and associates with several actin-binding proteins, including vinculin, we first studied the distribution of these proteins. Two cell populations

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can be distinguished in primary cultured CD cells: those located inside the colony and those located more peripherally in the epithelial cell sheets. In images from a middle confocal plane of untreated cultures, α-catenin and vinculin outlined the internal cells and colocalized at the cell–cell contacts (MOCControl 0.75±0.04) (Fig.2, quantitative analysis), thus reflecting the presence of AJ structures, as assessed in the segmentation images where colocalization appeared in white (Fig. 2A), and better observed in the enlarged figure of the indicated region (Fig. 2A'). In both internal and peripheral cells, the cytosolic pool of vinculin was also observed and when vinculin and α-catenin were not located in the plasma membrane, they did not colocalize (Fig. 2A-A'). When cultured cells were treated with increasing concentrations of D609, the typical epithelial morphology was progressively 10

ACCEPTED MANUSCRIPT altered (Fig. 2B-B', C-C', D-D'). By contrast to the high degree of vinculin–α-catenin colocalization in untreated cells, the SMS inhibitor induced an almost complete disappearance of colocalization between these proteins at cell–cell junctions, more noticeable at higher concentrations (MOC20μM 0.70±0.02, MOC50μM 0.64±0.01, MOC100μM 0.60±0.04; Control vs D609 100 μM, p<0.05) (Fig. 2B', C', D', segmentation images, and quantitative analysis). The cell contacts appeared disintegrated and most of the α-catenin

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dissipated from the lateral membrane, and appeared massively internalized as α-catenin-

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containing vesicle-like structures that also contained vinculin (Fig. 2B', C', asterisks). Some

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cells treated with 50 μM D609 appeared retracted, with the presence of α-catenin-positive bridges between adjacent cells (Fig. 2C', arrowheads). At the highest D609 concentration, cells showed an almost complete disintegration of cell-cell adhesion, with loss of the

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epithelial morphology (Fig. 2D-D'), and cell edges became irregular and acquired an elongated fibroblastoid-like morphology (Fig. 2D, arrows). However, despite the great

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deterioration, the cells remained attached to the ECM through FA (Fig. 2C'-D', arrows).

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Since besides interacting with the actin cytoskeleton, α-catenin binds β-catenin and through β-catenin binds E-cadherin to conform AJ, we further studied the distribution of these AJ

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proteins after the pharmacological inhibition of SMS. We treated cultured cells with 50 μM D609 because, at higher concentrations, the cells lost their epithelial phenotype, as shown

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in Fig. 2D-D'. In untreated cell cultures, β- and α-catenin delineated the cell borders, exhibiting a high degree of colocalization in the plasma membrane, as assessed in the

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segmentation images where colocalization appeared in white (Fig. 3A-A', segmentation). After D609 treatment, a thickening of the cell-cell junctions was observed in certain zones

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of the plasma membrane, reflecting weakening of the AJ (Fig. 3B, B', segmentation). When cell-cell adhesion was disrupted, α-/β–catenin-labeled filopodium-like structures were observed in the spaces between cells (Fig. 3B', arrows). Since the cytoplasmic E-cadherin domain binds β-catenin, which in turns binds α-catenin, thereafter, we analyzed the distribution of these proteins. As expected, in control cells, both proteins delineated the cell borders and colocalized, thus reflecting the presence of mature AJ (Fig. 3C-C', segmentation). After D609 treatment, the segmentation image shows a decrease in the degree of colocalization, and both proteins dissipated from the lateral membrane, and appeared massively internalized (Fig. 3D-D', segmentation), and β–catenin-labeled 11

ACCEPTED MANUSCRIPT filopodium-like structures were also observed in the spaces between cells (Fig. 3D'arrows). To confirm the effect of SMS inhibition on adhesion structures, α-catenin distribution was also evaluated in SMS 1 and SMS 2 knockdown cells. Positively transfected cells contained green dots in the cytosol, better observed in the phase contrast microscopy images (Fig. 4A-

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C). In scrambled siRNA-transfected cells, α-catenin delineated the cell contour (Fig. 4D),

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reflecting the integrity of cellular junctions. By contrast, SMS 1 siRNA-transfected cells presented a discontinuous α-catenin immunostaining in the plasma membrane,

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accompanied with irregular edges, thus reflecting the alteration of the cell-cell adhesion (Fig. 4E, arrows). SMS 2 siRNA-transfected cells showed no alterations in the plasma

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membrane α-catenin distribution (Fig. 4F). The overall alterations in SMS 1 but not in SMS 2 siRNA-transfected cells were also observed in the phase contrast microscopy images

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(Fig. 4A-C). Quantitative data showed that 88.2% of scrambled siRNA-transfected cells or 84.6 of SMS 2 siRNA-transfected cells had conserved phenotype while the 90.9 % of SMS

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1 siRNA-transfected cells presented altered phenotype. Taken together, these results suggest that SMS 1 but not SMS 2 activity is necessary to maintain intercellular adhesions

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in mature CD cells.

To evaluate the reversibility of the deleterious effect caused by the inhibition of SMS on

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cell-cell adhesions (Fig. 5A, B), cultured CD cells treated with 50 µM D609 were washed and re-incubated without the inhibitor for additional 24 h. The cells were immunostained

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with antibodies directed to vinculin and α-catenin. When the inhibitor was removed, cells recovered their cell-cell adhesions (Fig. 5C), thus denoting that when SMS activity is

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restored, cells re-establish the epithelial phenotype. Similar results were obteined when the cells were incubated with 50 µM D609 in the presence of 10 μM C12-SM exogenously aggregated (Fig.5D).

3.2 The inhibition of SMS 1 does not alter the delivery of E-cadherin at the cell surface E-cadherin molecules have a half-residence time of only several minutes in AJ, and are in continuous recycling [42]. Thus, the efficient delivery of E-cadherin to the lateral cell 12

ACCEPTED MANUSCRIPT membrane is critical for the preservation and functionality of AJ in polarized cells [43; 44]. Thereafter, we examined the surface level of E-cadherin by labeling the cell surface molecules with biotin, and examining the amount of labeled E-cadherin in control and D609-treated cells. To do so, after 72 h of culture, cells were incubated with D609 for 24 h. Then, cells were treated with the plasma membrane–impermeant biotinylating agent NHSSS-biotin before lysis. Biotinylated proteins were further precipitated with immobilized

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avidin. D609-treated cells showed an unexpected increased total and surface E-cadherin

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levels, when compared to untreated cells (Fig. 6A, Control vs D609, p=0.0193 and

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p<0.0024, respectively). It is known that to exhibit functional adhesion activity, E-cadherin has to form complexes with α- and β-catenin. Because β-catenin is a necessary intermediate in the linkage of α-catenin to the E-cadherin cytoplasmic domain, we performed Western

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blot analysis to investigate whether the D609-induced impairment in cell-cell adhesion was due to a decrease in the amount of these AJ proteins. Additionally, since vinculin, besides

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being a FA component, is also present at the cytoplasmic domains of cadherins, binding αand/or β-catenin [5, 13], we also analyzed vinculin levels. Surprisingly, the amount of

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vinculin and α-catenin levels increased (Fig. 6B, C vs D609, p<0.0001 and p<0.0008, respectively), whereas no significant change was observed in the amount of β-catenin (Fig.

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6B, C vs D609, pNS). This increased amount of vinculin and α-catenin probably correlates with the thick α-catenin immunostaining observed beneath the plasma membrane in D609-

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treated cells (Fig. 2B', C'). These findings suggest that the D609-induced impairment in intercellular adhesions was not due to a decrease in the amount of AJ proteins or to a

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decrease in the cell-surface level of E-cadherin.

3.3 Vinculin-immnostained FA increase after inhibition of SMS 1 activity The analysis of the immunofluorescence images showed that, although cell-cell adhesions were altered after D609 treatment, the cells remained attached to the ECM. It is known that during the first stage of tissue organization, FA formation precedes cell-cell junction [11]. The preservation of the cell-ECM attachment is a condition of survival, since epithelial cell-ECM detachment leads to cell death by anoikis [45, 46, 47, 48]. Therefore, we analyzed the effect of the inhibition of SMS activity on the preservation of cell-ECM adhesions. Since the number, size and distribution of FA can vary from one cell to another 13

ACCEPTED MANUSCRIPT or even within a single cell [49], we quantified the vinculin- and talin-immunostained FA in internal and peripheral cells of the colonies. Vinculin is a component of both FA and AJ, whereas– at least in CD cells- talin is present only in FA. For this reason, in untreated cells, vinculin but not talin, delineated cell contours (Fig. 7A, C). The quantitative analysis showed an unexpected significant increase in the number of vinculin-stained FA in both internal and peripheral D609-treated cells (Fig. 7B, and quantitative analysis). By contrast,

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the number of talin-stained FA in both internal and peripheral D609-treated cells was not

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altered (Fig. 7D, quantitative analysis. Although D609 treatment altered intercellular

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adhesions, as denoted by the formation of spaces between cells (Fig. 7B, D, arrows), cells

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remained attached to the ECM.

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3.4 The inhibition of SMS 1 induces a remodeling of the actin cytoskeleton During the tissue organization process, FA formation precedes AJ assembly. Vinculin acts as an actin-binding protein that connects the actin cytoskeleton inside the cell to AJ through

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its association to α-catenin. When it binds to AJ, actin organizes as a perijunctional actin

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belt [50; 51], whereas when it binds to FA through vinculin and talin, it organizes as stress fibers (SFs). Therefore, we analyzed the effect of SMS inhibition on actin cytoskeleton. As

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shown in FITC-phalloidin-stained untreated cells, actin appeared organized as SFs (Figs 8A and 9A, A') as well as a thin perijunctional actin belt, and actin arcs located in contact

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edges, where actin and vinculin colocalized (Fig. 8A, arrows and insert). This F-actin distribution corresponds to a correct cell organization of primary cultured cells, resembling

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tissue conformation. According to the nomenclature of SFs by Small [52], untreated inner cells show a predominance of ventral SFs, which associate with FA in both ends, whereas peripheral cells show a predominance of dorsal SFs, which associate with FA in one end of the fiber (Fig. 9A, A'). When cultured cells were treated with increasing concentrations of D609, the typical epithelial conformation was progressively altered as assessed by the dissipation of actin arcs accompanied by cell separation (Fig. 8B-D, inserts). This effect can be also observed in confocal images in Figure 9. At 20 μM, D609 altered cell-cell adhesion, and this alteration increased with the concentration of D609 (Fig. 9B´, C´, D- arrows). As D609 concentration increased, the progressive loss of epithelial morphology, resembling that of the migrating cell, was clearly seen in certain cells located in the periphery of the 14

ACCEPTED MANUSCRIPT colony (Fig. 8D, arrows). After D609 treatment, some changes in the SFs organization were observed. The increased alteration of cell-cell adhesion was accompanied with a predominance of ventral SFs associated with vinculin-stained FA (Figs 8D and 9B-D). The preservation of the actin organization in SFs can explain the maintenance of the spread morphology of the cells when intercellular junctions were altered. The appearance of a great number of vinculin-associated SFs correlates with the increased number of vinculin-

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stained FA observed in 50 μM D609-treated cells, previously reported (Fig. 7B,

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quantitative analysis).

3.5 The inhibition of SMS 1 induces a process of epithelial-mesenchymal transition

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(EMT) in CD cells

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One of the distinguishing features of the EMT process is the loss of the epithelial characteristics and the acquisition of properties typical of mesenchymal cells. To study the

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existence of a correlation between the morphological changes induced by the inhibitor D609 at 100 μM and the acquisition of the mesenchymal phenotype, we analyzed the

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expression of epithelial and mesenchymal markers. The immunocytochemical evaluation revealed that, in untreated primary cultures, CD cells were positive for the epithelial cell

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marker CK7 (Fig. 1C), and expressed α-catenin delimiting cell borders, typical of epithelial cells (Fig. 10A,I). By contrast, a low percentage of untreated cultured CD cells expressed

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vimentin, a mesenchymal cell marker, with a reticular pattern of immunostaining distribution (Fig. 10B-D, and 11A, quantitative analysis). Only some CD cells expressed α-

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SMA, a microfilament protein present in myofibroblasts considered as another mesenchymal cell marker (Figs 10J-L and 11A, quantitative analysis). In untreated conditions, we also observed a small number of overlapping cells with a fibroblast-like morphology, strongly positive for vimentin (Fig. 10B, arrows and 11B, quantitative analysis) and for α-SMA (Fig. 10J, arrows and 11B, quantitative analysis). After D609 treatment, an increase in the number of vimentin-positive CD cells was observed (Control vs D609, p=0.003) (Figs 10F-H and 11A, quantitative analysis). Notably, a great number of CD cells, which were almost negative in untreated conditions, expressed α-SMA after D609 treatment (Control vs D609, p=0.01) (Figs 10N-P and 11A, quantitative analysis), better observed in the enlargement shown in Fig. 10P. Moreover, the number of vimentin15

ACCEPTED MANUSCRIPT and α-SMA-positive overlapping cells was also increased (Control vs D609, vimentin and α-SMA: p=0.0001) (Fig. 10F, N-arrows, and 11B, quantitative analysis). To investigate the total level of both proteins, we further performed Western blot analysis. We found a correlation between the increased number of cells expresing vimentin and αSMA, and the level of both proteins when the cultured CD cells were treated with 100 μM

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D609 (Fig. 12, C vs D609, p=0.0132 and p=0.0002, respectively).

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The loss of intercellular adhesion, the actin cytoskeletal reorganization, and the acquisition

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of a fibroblastoid-like morphology, together with increased vimentin and α-SMA expression in D609-treated cultured CD cells led us to suggest that the inhibition of SMS 1

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could be inducing CD cells to undergo an EMT.

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4. Discussion

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In mammals, the ability of epithelial cells to join to each other and to the ECM is important in a high number of biological processes, including embryogenesis, wound healing and

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maintenance of tissue integrity and function. Sphingolipids, despite being a small proportion of membrane lipids, constitute a group of bioactive lipids whose participation in

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cellular processes is becoming more noticeable. SM, a major sphingolipid present in the plasma membrane, is highly enriched in lipid rafts. Studies from our laboratory have

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demonstrated that AJ and FA complexes are inserted in lipid rafts, and their phospholipid composition plays essential roles in the in vivo maintenance of cell adhesion structures in

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renal papillary CD cells [29, 30]. Using MDCK cells, we have also demonstrated that the intracellular synthesis of SM is a requirement for the acquisition of the differentiated epithelial cell phenotype [53]. In the present work, we studied the role of SMS activity in the maintenance of the differentiated epithelial phenotype of mature renal papillary CD cells. According to our results, the pharmacological inhibition of SMS and the knockdown of SMS 1 but not of SMS 2 altered cell-cell adhesions, thus indicating that SMS 1 but not SMS 2 is involved in the maintenance of tissue integrity. Cultured CD epithelial cells associated tightly with their neighbors, whereas D609-treated cells showed altered intercellular adhesions. It is accepted that cells require the constant expression and function of AJ proteins to remain associated and organized as an epithelium [54]. E-cadherin has a 16

ACCEPTED MANUSCRIPT half-residence time of only several minutes in AJ, and is in continuous recycling [42]. Thus, the efficient delivery of E-cadherin to the lateral cell membrane is critical for the preservation and funcionallity of AJ in polarized cells [1, 43, 44]. Different lines of evidences have demonstrated that recycling endosomes are rich in SM and cholesterol, and that SMS 1 is the main enzyme responsible for the generation of recycling endosomes [55, 56]. Consistently, in the present study, we observed that the expression of AJ proteins was

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not altered, as assessed by Western blot analysis of total CD cell homogenates. Moreover,

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the total intracellular amount of E-cadherin, α-catenin and vinculin was increased,

amount of proteins that are part of the AJ structure.

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indicating that the observed alterations in cell-cell adhesions were not due to a decreased

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Li et al. reported that the down-regulation of the two SMS isoforms alters the amount of SM exclusively in the plasma membrane raft domains of HEK 293 cells [57]. As SMS 1

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resides in the Golgi apparatus, it has long been suggested that similar raft-like domains exist in the Golgi membranes [58, 59, 60]. These observations, together with our present

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results, suggest that the inhibition of SMS 1 could induce modifications in the lipid composition of the vesicles that transport E-cadherin through the plasma membrane,

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creating a different lipid environment where E-cadherin inserts. Moreover, E-cadherin is synthesized as a precursor and then suffers multiple post-translational modifications which

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regulate E-cadherin transport, membrane assembly and interaction with E-cadherin dimers in adjacent cells [61, 62]. This processing is essential for E-cadherin maturation and cell

[63, 64].

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adhesion; and the lateral dimerization of E-cadherin is required for its adhesive function

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In the present study, we showed that the D609-induced impairment of cell-cell adhesion was accompainied by an increase in the number of vinculin- but not talin-stained FA, with the consequent increase in ventral SFs, thus assuring the cell-ECM attachment. FA are hierarchical structures which, during maturation, evoke the sequential binding of several proteins such as talin, vinculin, and paxillin, thus allowing the assembly and disassembly of FA [8], and constitute the remodeling process that gives plasticity to the cells. It is acepted that during the first stage of tissue organization, FA formation precedes cell-cell junction [11]. Interestingly, the inhibition of SMS activity caused an increase in the number of FA containing vinculin. So, the inhibition of SMS activity could be inducing a return to the 17

ACCEPTED MANUSCRIPT early stages of tissue organization, to keep cell adhesion to the ECM, thus avoiding anoikis [45, 46, 47, 48]. Taken together, we can speculate that the preservation of CD cell attachment to the ECM probably allow the further regeneration of the epithelial sheet (tubular epithelium), as shown in the experiments where by washing the inhibitor, cells recovered their ability to form AJ, probably because the transport of the AJ complex was

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re-established. In this context, our results could be of physiological relevance.

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Cultured CD cells expressed the epithelial cell marker CK7, whereas only some cells expressed vimentin, and few CD cells lightly expressed α-SMA, resembling the epithelial

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phenotype of CD cells. Vimentin, besides being the main intermediate filament protein of mesenchymal cells, is frequently co-expressed with other members of the intermediate

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filaments such as cytokeratins [65]. Grupp et al. reported that CD cells become positive for vimentin in cell culture, and thus concluded that vimentin staining is not reliable for the

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discrimination between epithelial and mesenchymal cells in renal tissue [66]. In untreated conditions, our cultured CD cells were almost negative for α-SMA, but after D609

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treatment, cells became intensively positive for α-SMA, denoting de novo synthesis of the protein. Moreover, the number of vimentin-stained CD cells and that of vimentin- and α-

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SMA-positive overlapping cells were also increased. α-SMA, an actin isoform typical of smooth muscle cells, is present in the cytoplasm of myofibroblasts [67], and is considered a

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mesenchymal cell marker [68, 69]. In adult tissues, myofibroblasts are involved in wound healing and become activated by inflammation [70], and like fibroblasts, they have a great

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ability to produce ECM components [71, 72]. In fact, myofibroblasts are considered activated fibroblasts [73]. In the present study, we showed that SM synthesis inhibition

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induced a loss of intercellular adhesion, actin cytoskeletal reorganization, and the acquisition of a fibloblast-like morphology more notably observed at higher concentrations of the SMS inhibitor. These features are consistent with the alterations in morphology and cellular architecture reported in tubular epithelial cells during renal fibrosis [33, 74]. The EMT, a form of cell plasticity in which differentiated epithelial cells acquire mesenchymal phenotypes, is increasingly recognized as an integral aspect of tissue fibrogenesis [33, 36, 74]. α-SMA is especially present in myofibroblasts which represent an advanced stage of EMT associated with fibrosis [67, 75]. It has been reported that a line of CD cells could undergo an EMT in vitro after stimulation with IGF-I and TGF-1 [76]. We 18

ACCEPTED MANUSCRIPT interpreted that the low percentage of α-SMA/vimentin-positive cells observed in untreated culture represents cells that evoke EMT in basal conditions. The presence of these overlapping cells could be attributed to the fact that EMT in the adult is regarded as a physiological attempt to repair damaged tissue and to control inflammation [35] since, as we have previously reported, the mitotic index in renal papillae of adult rats is very low

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[77].

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Taking into account the changes observed in CD cell morphology, together with the increase in vimentin expression and de novo synthesis of α-SMA by CD cells, we suggest

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that the inhibition of SMS 1 activity induces CD cells to undergo an EMT and that the new overlying myofibroblasts may thus be derived from pre-existing CD cells. In our opinion,

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by inhibiting SMS 1 activity, the balance between the EMT-MET processes is altered, generating new mesenchymal cells because of the difficulty to form new AJ. In this

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context, besides proposing for the first time that SM synthesis is an important tool for maintaining the balance between EMT-MET, we also propose SMS 1 as a modulator of this

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process. It appears that, by inhibiting SMS 1 activity, we are not triggering a new process, but rather altering the balance of an existing one, which is the main phenomenon by which

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diseases occur. Moreover, it has been widely reported that normal aging of the kidney is associated with mild forms of fibrosis, which suggests that, as ageing occurs, the normal

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injury-repair cycle becomes impaired [78]. The occurrence of specific diseases related to

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5. Conclusion

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the lack of activity of this enzyme in renal tissue requires further studies.

In the present study, we demonstrated that the preservation of mature AJ, and therefore of epithelial tissue organization, requires active intracellular activity of SMS 1, and we also demonstrated that when this activity is inhibited, cells progress to the acquisition of a mesenchymal phenotype.

19

ACCEPTED MANUSCRIPT Acknowledgements - Mr Roberto Fernández for confocal microscope technical assistance. This work was supported by the National Council for Scientific and Technologic ResearchCONICET (PIP-0233 and PIP-502), by National Agency for Scientific and Technologic Promotion (PICT-1038), by University of Buenos Aires and by the National University of

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La Rioja (27/A626 and 27/A674).

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[69] W.C.C. Lee, J.P. Rubin, K.G. Marra, Regulation of α-smooth muscle actin protein expression in adipose-derived stem cells, Cells Tissues Organs 183 (2006) 60-86. [70] B. Eckes, E. Colucci-Guyon, H. Smola, S. Nodder, C. Babinet, T. Krieg, P. Martin, Impaired wound healing in embryonic and adult mice lacking vimentin, J. Cell Sci. 113 (2000) 2455-2462. [71] A. Desmoulière, I.A. Darby, G. Gabbiani, Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis, Lab. Invest. 83(12) (2003) 16891707. [72] M. Guarino, A. Tosoni, M. Nebuloni, Direct contribution of epithelium to organ fibrosis: epithelial-mesenchymal transition, Hum. Pathol. 40 (2009) 1365–1376. 24

ACCEPTED MANUSCRIPT [73] D.W. Powell, I.V. Pinchuk, J.I. Saada, X. Chen, R.C. Mifflin, Mesenchymal cells of the intestinal lamina propria, Annu. Rev. Physiol. 73 (2011) 213–237. [74] Y. Liu, Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention, J. Am. Soc. Nephrol. 15 (2004) 1–12. [75] A. Masszi, C. Di Ciano, G. Sirokmány, W.T. Arthur, O.D. Rotstein, J. Wang, C.A.G. McCulloch, L. Rosivall, I. Mucsi, A. Kapus, Central role for Rho in TGF-1-induced -smooth muscle actin expression during epithelial-mesenchymal transition, Am. J. Physiol. - Renal Physiol. 284 (2003)

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[76] L. Ivanova, M.J. Butt, D.G. Matsell, Mesenchymal transition in kidney collecting duct epithelial

[77] M.G. Márquez, I. Cabrera, D.J. Serrano, N.B. Sterin-Speziale, Cell proliferation and morphometric changes in the rat kidney during postnatal development, Anat. Embriol. 205 (2002) 431-440.

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[78] B.L. Kasiske, Relationship between vascular disease and age-associated changes in the human

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kidney. Kidney Int. 31(5) (1987) 1153-1159.

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ACCEPTED MANUSCRIPT Figure legends Figure 1: Collecting duct cells preserve the fully differentiated epithelial phenotype when cultured. (A) Phase contrast image of a 96 h primary culture of CD cells without treatment. (B) Wide-field fluorescence microscopy showing the presence of the primary cilium immunostained with an anti-acetylated tubulin antibody, and (C) the expression of the epithelial cell marker CK7. (B') shows a magnification of the region indicated in B to

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simplify the visualization of the primary cilium (arrows). Nuclei were counterstained with

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of three different experiments are shown. Scale bar: 50 μm.

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Hoechst 33258 (blue). Images from diferent microscopic fields and representative images

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Figure 2: Effect of increasing concentrations of D609 on cell-cell adhesion structures. Cultured CD cells were incubated in the presence of increasing concentrations of D609, a

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pharmacological inhibitor of SMS 1 and 2, and then double-immunolabeled with antivinculin (green) and anti-α-catenin (red) antibodies. The second column of images (A´-D´)

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shows the enlargements of the areas indicated in the boxes, whereas the third column

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corresponds to the segmentation images of the non-enlarged figures, which indicate the colocalization of proteins in white on a black background. Asterisks in B' and C' show internalized α-catenin- and vinculin-containing vesicle-like structures. Arrowheads in C'

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show α-catenin-positive bridges between adjacent cells. Arrows in C' and D' show FA, and in D, cells with fibroblastoid-like morphology. Confocal microscopy images are shown.

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Scale bar: 20 μm.

Figure 3: Effect of pharmacological inhibition of SMS in cell-cell adhesions. Cultured CD cells were incubated in the presence of D609 at the concentration of 50 μM, and then double-immunolabeled with anti-α-catenin (red) and β-catenin (green) antibodies (A-B) or anti-β-catenin (green) and E-cadherin (red) antibodies (C-D). The nuclei were stained with Hoechst 33258 (blue). The second column of images shows the enlargements of the areas indicated in boxes, whereas the third column corresponds to the segmentation images of the non-enlarged figures, which indicate the colocalization of proteins in white on a black

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ACCEPTED MANUSCRIPT background. Arrows in B' and D' show filopodia-like structures in the spaces between cells. Wide-field fluorescence microscopy images are shown. Scale bar: 50 μm.

Figure 4: SMS 1 but not SMS 2 knockdown impairs cell-cell adhesions. Cultured CD cells were incubated with scrambled siRNA (A and D), SMS 1 siRNA (B and E), or SMS 2

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siRNA (C and F). (A-C) Show phase contrast microscopy images. Green dots indicate

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positive transfected cells. (D-F) show cultured cells immunostained with an anti-α-catenin

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antibody to study intercellular adhesions. Nuclei were counterstained with Hoechst 33258 (blue). Arrows in E point alterations in cell-cell adhesion. Representative images of three

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experiments are shown. Scale bar: 50 µm.

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Figure 5: The effect of the inhibition of SMS on intercellular adhesions is reversible. Cultured CD cells were incubated with D609 at 50 μM (B), and after washing, the cells

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were re-incubated for additional 24 h in the absence of D609 (C). Cultured CD cells were incubated with D609 at 50 μM with exogenous 10 μM C12-SM for 24 h (D). Then, the

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cells were double-immunolabeled with anti-α-catenin (red) and vinculin (green) antibodies. (A) Control cells show vinculin and α-catenin colocalization in cell edges, reflecting the

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integrity of adherens junctions (insert). (B) After D609 treatment, the cells show spaces between them (arrowheads and insert). (C) After re-incubation without the inhibitor, or (D)

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with D609 in the presence of exogenous C12-SM, cells restored the intercellular adhesions, where both proteins colocalized (inserts). Nuclei were counterstained with Hoechst 33258

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(blue). Representative images of three experiments are shown. Scale bar: 50 µm.

Figure 6: Effect of the inhibition of SMS activity on the E-cadherin delivery to the cell surface. Cultured CD cells were incubated with 50 μM D609 and processed as described in Materials and methods. (A) Surface E-cadherins were biotinylated. Cells were lysed and the biotinylated proteins were recovered with streptavidin beads. Surface E-cadherin of wholecell lysates was analyzed by Western blot with E-cadherin antibody. Results are expressed as a percentage of the control (hypothetical value of 100%) and correspond to the mean ± 27

ACCEPTED MANUSCRIPT SEM of three independent experiments. (B) Analysis of the total amount of adherens junction proteins. To perform the Western blot, equal amounts of total proteins were seeded in each experimental condition and antibodies directed against the indicated proteins were used. Results are expressed as a percentage of the control (hypothetical value of 100%) and correspond to the mean ± SEM of three independent experiments. (*) Significant

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Figure 7: Effect of the inhibition of SMS activity on FA. Cultured CD cells were incubated with 50 μM D609, and immunostained with anti-vinculin (A,B) and anti-talin (C,D) antibodies. Wide-field fluorescence microscopy showing the basal area of the cells,

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where FA are located. Arrows in B and D indicate cells in the process of separation. Scale bar: 50 µm. Quantitative analysis of vinculin- and talin-stained FA. The number of FA per

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cell in the different areas of the colonies was calculated by an image analysis program, as described in Materials and methods. The results correspond to the mean ± SEM of three

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experiments. (*) Significant differences with respect to control.

Figure 8: Effect of the inhibition of SMS activity on the actin cytoskeleton. Cultured

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CD cells were incubated with increasing concentrations of D609 and immunolabeled with antibodies directed against vinculin (red) to detect FA and AJ, and phalloidin-FITC (green)

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to label the actin filaments. Nuclei were stained with Hoechst 33258 (blue). Wide-field fluorescence microscopy images are shown. The inserts show amplifications of some

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regions of interest, where the loss of the colocalization (yellow) of the proteins in the circumferential belt is better observed. The arrow in C indicates a cell in the process of separation, and in D, cells that have lost the epithelial morphology. Representative images of three experiments are shown. Scale bar: 50 μm.

Figure 9: Effect of the inhibition of SMS activity on stress fibers. Cultured CD cells were incubated with increasing concentrations of D609 and immunolabeled with vinculin antibody (red) to detect FA and phalloidin-FITC (green) to label the actin filaments. 28

ACCEPTED MANUSCRIPT Confocal microscopy images captured with a 60X objective are shown. The second column of images shows the enlargements of the areas indicated in boxes. Arrows in B´, C´, and D indicate cells in the process of separation. Arrowheads in A´, B´, C´ and D´ indicate FA. Representative images of three independent experiments are shown. Scale bar: 20 μM.

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Figure 10: Effect of the inhibition of SMS activity on the expression of mesenchymal

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proteins. Cultured CD cells were incubated with 100 μM D609, and immunostained with

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anti-α-catenin (red), anti-vimentin (green), and α-SMA (green) antibodies. Nuclei were stained with Hoechst 33258 (blue). Arrows indicate overlapping cells with a fibroblast-like morphology. D,H,L and P show the enlargements of the areas indicated in boxes.

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Representative images of three independent experiments are shown. Scale bar: 50 μM.

Figure 11: Quantitative analysis of vimentin- and α-SMA-immunostained CD cells.

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(A) Number of vimentin- and α-SMA-positive CD cells expressed as percentage (mean ± SEM). Unpaired t test with Welch correction: vimentin: p=0.003, and α-SMA: p=0.01. (B)

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Number of vimentin- and α-SMA-positive overlapping cells expressed as positive cells found per microscopic field (mean ± SEM). Unpaired t test: vimentin and α-SMA:

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p=0.0001. (*) Significant differences with respect to control.

Figure 12: Analysis of the total amount of vimentin and α-SMA. Cultured CD cells

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were incubated with 100 μM D609 and processed as described in Materials and methods. To perform the Western blot, equal amounts of total proteins were seeded in each experimental condition and antibodies directed against the indicated proteins were used. Results are expressed as a percentage of the control (hypothetical value of 100%) and correspond to the mean ± SEM of three independent experiments. (*) Significant differences with respect to control.

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ACCEPTED MANUSCRIPT Highlights

Sphingomyelin synthase (SMS) 1 activity is essential for adherens junctions maintenance.

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Contrary SMS 1 activity is not necessary for focal adhesions (FA) maintenance.

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The inhibition of sphingomyelin synthase induce the loss of epithelial phenotype

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This enzyme inhibition proposes a novel model of epithelial–mesenchymal transition

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modulation.

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