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Vol. 17, Issue 8, 3435-3445, August 2006
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*Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, 28006 Madrid, Spain; Centro Nacional de Investigaciones Cardiovasculares (CNIC), Unidad Mixta CNIC-Universitat de Valencia, 46010 Valencia, Spain; Chemical Biology Program, The Broad Institute of Harvard University and Massachusetts Institute of Technology, Cambridge, MA 02141; and ||Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
Submitted January 4, 2006;
Revised May 17, 2006;
Accepted May 18, 2006
Monitoring Editor: Martin A. Schwartz
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Chemokines are a family of low-molecular-weight cytokines that bind to G protein–coupled receptors on the cell surface (Mackay, 2001). In addition to their classical function as chemoattractants, chemokines modulate lymphocyte adhesion to endothelium, through a yet-poorly understood mechanism of receptor cross-talk between chemotactic and adhesion receptors (Alon and Feigelson, 2002; von Andrian and Mackay, 2000). Chemokines also trigger the remodeling of cytoskeleton and the reorganization of multiple plasma membrane receptors and signaling molecules, which result in an overall change of lymphocyte shape and the acquisition of a migratory, polarized morphology (Sanchez-Madrid and del Pozo, 1999). Lymphocyte polarization involves the generation of two well-differentiated poles. The leading edge clusters actin microfilaments, actin-associated proteins, and signaling molecules that generate protrusive structures, and concentrates adhesion receptors at the cell front. On the other hand, adhesions are released at the rear trailing edge to enable net cell movement (Serrador et al., 1999; Worthylake and Burridge, 2001; Etienne-Manneville and Hall, 2002; Ridley et al., 2003; Vicente-Manzanares and Sanchez-Madrid, 2004).
Although there is much knowledge on how the actin cytoskeleton participates in cell migration (Pantaloni et al., 2001; Pollard and Borisy, 2003), the role played by tubulin and associated proteins in this process remains controversial and seems cell type-dependent (Wittmann and Waterman-Storer, 2001; Destaing et al., 2005). However, it is clear that, in addition to their role during cell division, microtubules play important functions in the establishment and maintenance of cell polarity and adhesion (Prescott et al., 1989; Tannenbaum and Slepecky, 1997; Moreno and Schatten, 2000; Small and Kaverina, 2003). The majority of interphase microtubules are dynamic, but a small proportion is relatively stable (Schulze and Kirschner, 1987), showing a low rate of depolymerization by colchicine or cold. Microtubule stability is related to post-translational modifications of tubulin subunits (Gundersen et al., 1998), including the acetylation of the Lys40 
-amino group of 
-tubulin (L’Hernault and Rosenbaum, 1983; Piperno and Fuller, 1985). This type of acetylation has been found in a variety of proteins (transcription and nuclear import factors, histones) and is catalyzed by a wide range of acetyltransferases (Kouzarides, 2000; Polevoda and Sherman, 2002). In cultured cells, acetylation seems to preferentially occur in polymerized microtubules (Piperno et al., 1987). It has been shown that in vivo, deacetylation takes place on tubulin dimers, rather than microtubules (Matsuyama et al., 2002).
HDAC6, a class II histone deacetylase mainly localized in the cytoplasm (Verdel et al., 2000; Bertos et al., 2004), has two catalytic domains with deacetylase activity. The C-terminal domain is able to deacetylate -tubulin both in vitro and in vivo, and this activity is reversibly inhibited by trichostatin A (TSA) and tubacin. TSA is a broad HDACs inhibitor, whereas tubacin is specific for the tubulin deacetylase activity of HDAC6 (Hubbert et al., 2002; Matsuyama et al., 2002; Haggarty et al., 2003; Zhang et al., 2003). These compounds inhibit the HDAC6 deacetylase activity by chelating a Zn2+ cation (Furumai et al., 2001; Jose et al., 2004) and may also alter the formation of dynamic molecular complexes of HDAC6 with other intracellular proteins such PP1, HSP90, Dia2, dynein, or tubulin (Kawaguchi et al., 2003; Brush et al., 2004; Destaing et al., 2005; Hideshima et al., 2005; Kovacs et al., 2005). In this regard, it has been reported that TSA disrupts HDAC6/PP1 association, affecting the enzymatic activity of both molecules (Brush et al., 2004).
We have herein investigated the role of HDAC6 in the migration of T-lymphocytes. We found that HDAC6 is mainly concentrated at cellular regions with high protrusive activity in migrating lymphocytes and that its expression modulates lymphocyte chemotaxis independently of its enzymatic activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was from R&D Systems (Minneapolis, MN), and sodium butyrate from Sigma Chemical Co. (St. Louis, MO). Tubacin, DHM-tubacin, niltubacin, MAZ-1391, MAZ-1338, and MAZ-1380 were synthesized according to Mazitschek et al. (unpublished data). The 80-kDa fibronectin fragment (FN80) was a generous gift from Dr. A. García-Pardo (Centro de Investigaciones Biológicas, Madrid, Spain). Phytohemaglutinin A (PHA) and interleukin-2 (IL-2) were from Sigma. Rabbit and goat anti-human HDAC6 polyclonal antibodies were purchased from MBL (Watertown, MA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. The anti--tubulin B-5–1-2 monoclonal antibody (mAb), the FITC-conjugated anti--tubulin (clone DM1A), and the anti-acetylated -tubulin 6–11B-1 mAbs were purchased from Sigma. The JL-8 anti-GFP mAb was from BD Biosciences Clontech (Palo Alto, CA). For surface molecule staining, the following mAbs were used: HP2/19 anti-ICAM-3, 12G5 anti CXCR4, MAB181 anti-CCR5, PL-1 anti-PSGL-1, anti-CD62L, Lia3/2 anti-CD18, HP2/21 anti-CD43 and HUTS-21 anti-activated 
1 integrins.
Cells
Human T-cell lines HSB-2 and CEM 1.3 were grown in RPMI 1640 culture medium (Invitrogen, Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS). Human peripheral blood lymphocytes (PBLs) were obtained as described by Campanero et al. (1994), and T lymphoblasts by 48-h treatment with 1 µg/ml PHA, followed by 50 U/ml IL-2 in RPMI 1640 medium until the eleventh day.
Transfection of Cells and Recombinant DNA Constructs
PBLs were washed once in phosphate-buffered saline and resuspended (1.2 x 107 cells/ml) in electroporation buffer containing 12 µg of plasmid DNA pEGFP, wtHDAC6-EGFP or double mutant HDAC6 H216A/H611A-EGFP (HDAC6 DD). Cell suspensions (100 µL) were transferred to a 2.0-mm electroporation cuvette and nucleofected with an Amaxa Nucleofector apparatus (Amaxa GmbH, Cologne, Germany). Then, cells were transferred to complete medium without antibiotic and cultured in six-well plates at 37°C until analysis. HSB-2 cells were transfected by electroporation. The human T-cell line CEM 1.3 was transduced with the retroviral vector pLZR IRES to stably express EGFP, wtHDAC6-EGFP, or HDAC6 H216A/H611A-EGFP. Retroviruses were produced by transfection of the Phoenix packaging cell line with a DNA mixture containing 2.5 µg env (pVSV-G; Clontech, BD Biosciences), 4 µg gag-pol (pNGVL3-MLV) and 3.5 µg retroviral vector pLZR IRES (a generous gift from Dr. A. Bernad, Centro Nacional de Biotecnología, Cantoblanco, Madrid, Spain). Supernatant with retroviruses was recovered and filtered 48 h after transfection and diluted 1:2 in RPMI 1640 medium. The infection was carried out by spinning 5.0 x 105 CEM cells, with 200 µl of retroviral supernatant and polybrene at 6 µg/ml, per well (24-well plates, Costar, Corning, NY), at 1800 rpm, 30°C for 90 min. Finally, 300 µl of complete RPMI medium was added, and cells were cultured at 37°C in 5% CO2 atmosphere.
siRNA Assay
Double-stranded siRNA (21 pb) against the HDAC6 217–237 (Hubbert et al., 2002; Serrador et al., 2004) and 284–304 sequences, and the negative control was purchased from Eurogentec (Hampshire, United Kingdom). PBL cells (7.0 x 106) were nucleofected with the siRNA using the Human T-Cell nucleofector kit (Amaxa GmbH). Efficiency was assessed by Western blot.
Immunofluorescence Microscopy
These assays were performed as described (Serrador et al., 2004). Briefly, 5.0 x 105 cells were allowed to adhere to coverslips coated with FN80. When indicated, cells were pretreated or not with SDF-1, TSA, TSA+SDF-1, NCD, TXL, or sodium butyrate. Cells were then fixed and stained for acetylated and total -tubulin using the 6-11B-1 and DM1A antibodies. Endogenous HDAC6 was stained with the goat anti-HDAC6 antibody and a secondary donkey anti-goat Alexa-488–conjugated antibody. Cells were observed in a Leica DMR photomicroscope (Leica, Mannheim, Germany), and images were visualized, processed, and stored by using the Leica QFISH software.
Time-Lapse Fluorescence Confocal Microscopy
PBL or CEM cells (5 x 105) were allowed to adhere on FN-coated coverslips, maintained in 1 ml of 2% BSA Hanks' balanced salt solution (HBSS) on Attofluor open chambers (Molecular Probes, Eugene, OR), and placed on the microscope stage. Cells were maintained at 37°C in a 5% CO2 atmosphere. Series of fluorescence and differential interference contrast frames were obtained simultaneously, using a Leica TCS SP confocal laser scanning unit attached to a Leica DMIRBE inverted epifluorescence microscope.
Chemotaxis Assay in Transwell Chambers
Assays for lymphocyte chemotaxis were performed in nude or TNF-–activated human umbilical vein endothelial cells (HUVEC)-coated polycarbonate membranes of 6.5-mm diameter, 10-µm thickness, and 3- or 5-µm-diameter pore size Transwell chambers (Costar). Lymphocytes (100 µl at 1.0 x 106 cells/ml) resuspended in 0.5% human serum albumin RPMI 1640 were added to the upper chamber. Then, chemokines (100 ng/ml SDF-1) were added to the lower chamber, and cells were allowed to migrate for 2 h at 37°C in 5% CO2 atmosphere. Then, the migrated cells were recovered and counted by flow cytometry.
Transendothelial Migration under Flow Conditions
The parallel plate flow chamber used for leukocyte adhesion and transmigration under defined laminar flow has been described (Barreiro et al., 2002). PBL or CEM T-cells (106/ml) were drawn on a TNF-–activated confluent endothelial monolayer at an estimated wall shear stress of 1.8 dyn/cm2 for a total perfusion time of 10 min. Lymphocyte rolling on the endothelium was easily visualized because they traveled more slowly than free-flowing cells. Lymphocytes were considered to be adherent after 20 s of stable contact with the cell monolayer. Transmigrated lymphocytes were determined as being beneath the endothelial monolayer. Lymphocytes were considered detached when they returned to free-flowing after their complete arrest on endothelium. The number of rolling, adhered, transmigrated, and detached cells was quantified by direct visualization of eight different fields (20x phase-contrast objective) at each time point of every independent experiment.
For detachment experiments, PBLs untreated or pretreated with TSA or sodium butyrate for 1 h were allowed to adhere for 4 min at 37°C to activated HUVEC monolayers. Then, shear stress was applied by pulling assay buffer (HBSS buffer with 2% FCS) through the flow chamber with a calibrated programmable pump, starting at 0.5 dyn/cm2 and increasing up to 10 dyn/cm2 at 1-min intervals. Attached and transmigrated cells after each shear stress interval were quantified (20x phase-contrast objective), and the mean ± SD of seven fields is presented. Cell detachment was obtained from the difference of final to initial adhered plus transmigrated cells (Barreiro et al., 2005).
Western Blot Analysis
Cell lysates were subjected to 10% SDS-PAGE under reducing conditions, and transferred onto nitrocellulose membranes (Trans-Blot, Bio-Rad, Hercules, CA). Then, membranes were saturated with tris-buffered saline-0.1% Tween containing 3% BSA and incubated with mAb against -tubulin or acetylated--tubulin or HDAC6 or GFP, followed by a peroxidase-conjugated secondary antibody. Proteins were visualized by enhanced chemiluminescence, and densitometric analyses were performed with ImageGauge 3.46 software (Fuji Photo Film Co., Tokyo, Japan).
Tubacin-niltubacin competition assays were carried out for 120 min at 37°C and 5% CO2 in CEM leukemic T-cells and PBLs. Tubacin (0.25 or 0.5 µM) was coincubated with at least eight different concentrations, from 1 nM to10 µM, of niltubacin to determine IC50 (concentration [M] of this competing ligand that inhibits by 50% the effect of tubacin binding). Western blotting of acetylated tubulin resulting from the competitive effect of niltubacin versus tubacin for HDAC6 was then performed. Data were analyzed using GraphPad Prism 2.0 software (GraphPad Software, San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), also displayed significant amounts of acetylated -tubulin concentrated at their uropods (Figure 1B).
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). When TSA-treated PBLs were induced to polarize with SDF-1, they acquired a polarized migratory morphology, and HDAC6 was redistributed to the leading edge and the uropod (Figure 2, C and D). Live-cell time-lapse confocal videomicroscopy confirmed the localization of HDAC6 at motility-associated structures in PBLs transfected with HDAC6-GFP and stimulated with SDF-1 (Figure 2D and sal material, Supplementary Video 1).
Lymphocyte Transmigration Inhibition by HDAC6 Inhibitor TSA
We then investigated the role of HDAC6 in lymphocyte migration. Pretreatment of PBLs with TSA for 1 h (to avoid its effect on gene transcription) decreased lymphocyte chemotaxis and their transendothelial migration (Figure 3A, and B). Similar results were observed in HSB-2 and CEM T-cells (unpublished data). The inhibitory effect of TSA was more evident when lymphocytes were allowed to transmigrate across the endothelial cell monolayer. On the other hand, treatment of lymphocytes with TXL decreased cell migration, whereas NCD had an opposing effect (Figure 3B). Additional experiments showed that TSA did not have a significant effect on T-cell adhesion or the expression of chemokine and adhesion receptors (Figure 3, C and D, and Table 1A).
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).
Deacetylase Activity of HDAC6 Is Not Involved in Lymphocyte Migration
To further explore the role of HDAC6 enzymatic activity in lymphocyte migration, we used tubacin (a specific HDAC6 tubulin deacetylase inhibitor), and other deacetylase inactive tubacin derivatives (niltubacin, MAZ-1391, MAZ-1338, and MAZ-TBDPS-O-1380) (Figure 4, A and B). Tubacin inhibits HDAC6 tubulin deacetylase activity in a dose-dependent manner, reaching a plateau at 10–20 µM, whereas niltubacin did not exert any effect at the same doses (Figure 4B and unpublished data). Tubacin inhibited the chemotaxis of PBLs, HSB-2, and CEM T-cells induced by SDF-1 (Figure 4C and unpublished data). Unexpectedly, the inactive compounds niltubacin, MAZ-1338, and MAZ-1391 also decreased cell migration (Figure 4C). In contrast, the inactive derivative MAZ-TBDPS-O-1380 did not exert this effect (Figure 4C). Neither tubacin nor niltubacin affected the adhesion of PBLs or T-cells to FN-80 in static conditions (Figure 4D). Likewise, no significant changes on cell viability and the expression levels of different adhesion and chemokine receptors were detected when CEM cells were treated with tubacin or niltubacin (Table 1B and unpublished data).
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, similar results were observed in cells treated with TSA, tubacin or niltubacin, which suggest that HDAC6 tubulin deacetylase activity is neither essential for lymphocyte polarization (Figures 2 and 6) nor migration (Figures 4C and 5).
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(Figure 8, B–D). To unequivocally demonstrate that HDAC6 molecule itself was accounting for the above-described functional effects, HDAC6wt, and catalytically inactive HDAC6DD were compared in restoring the migration of HDAC6-interfered cells. The results convincingly show that defective cell migration is reverted at comparable level by overexpression of both HDAC6wt and HDAC6DD molecules (Figure 8C) and that the effect is HDAC6 specific. Moreover, adhesion and transmigration of HDAC6 knockdown CEM cells to activated HUVEC was assessed under shear stress conditions. As observed above for TSA-treated lymphocytes (Figure 3, E and F), a reduced number of adherent and transmigrated lymphocytes was found in HDAC6-knockdown cells (Figure 8D). These data show that the overexpression of HDAC6 enhances lymphocyte migration, whereas its targeting inhibits lymphocyte chemotaxis. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Terzis et al., 1997; Westerlund et al., 1997; Hubbert et al., 2002; Haggarty et al., 2003), little is known regarding its involvement in the motility of leukocytes. In this work, we have addressed the possible role of HDAC6 tubulin deacetylase in the migratory activity of T-lymphocytes. We have found that activated T-cells show increased levels of acetylated microtubules around the MTOC, partially colocalizing with HDAC6. It has been reported that the microtubular network is retracted toward the uropod in T-cells polarized by chemokines (Ratner et al., 1997). On the other hand, we and others have previously shown that microtubules do not play an important role in uropod formation (Wilkinson, 1986; Campanero et al., 1994; Brown et al., 2001). Whether the enhanced levels of acetylated microtubules seen in polarized migrating T-lymphocytes are part of the mechanism by which microtubular bundles are retracted into the uropod or occur after retraction of the microtubule meshwork into this structure is still an open question. However, our data using different chemical inhibitors of HDAC6 and experiments with cells overexpressing HDAC6 deacetylase dead mutant do not support a role for tubulin deacetylase activity in the acquisition of a polarized migratory cell shape.
Our data on the localization of HDAC6 at motile protrusive structures, such as the leading edge and the uropod, support a possible involvement of this molecule in the regulation of lymphocyte locomotion. Accordingly, we found that the migration of T-cells in response to chemokines is reduced by HDAC6 siRNA and increased by HDAC6 overexpression and that these effects are not due to changes in cell adhesiveness under static conditions. Nevertheless, our data under shear stress conditions clearly indicate that adhesion strength is weaker in lymphocytes in which HDAC6 has been targeted by either a chemical inhibitor or specific siRNA knockdown. Hence, HDAC6 molecules appear to be required for adhesion and transmigration of lymphocytes under mechanical stress conditions or when cells transmigrate across constricted spaces. These data, together with similar results in mouse NIH3T3 fibroblasts (Hubbert et al., 2002), suggest that HDAC6 levels modify molecular interactions or intracellular signaling pathways involved in the modulation of cell motility. Interestingly, we have found that the levels of HDAC6 but not its deacetylase activity seems to be critical for lymphocyte chemotaxis. Hence, our data with a panel of tubacin derivatives, together with the results with cells overexpressing wt and deacetylase inactive forms of HDAC6, underscore the critical role of this molecule in lymphocyte motility regardless of its enzymatic activity. This is apparently in disagreement with data obtained in NIH3T3 fibroblasts, in which HDAC6 deacetylase activity was related to cell motility (Hubbert et al., 2002; Haggarty et al., 2003; Palazzo et al., 2003). However, it is feasible that HDAC6 has a scaffold role for the rapid formation of molecular complexes required for lymphocyte migration, a process 10- to 20-fold faster than that of fibroblasts (Serrador et al., 1999). Hence, very dynamic processes such as leukocyte locomotion would require a rapid HDAC6-mediated on/off assembly of molecular complexes, whereas this effect of HDAC6 would be dispensable in slow dynamic processes such as fibroblast locomotion or other phenomena involving cell-to-cell immune or viral-mediated interactions (Serrador et al., 2004; Valenzuela-Fernandez et al., 2005).
Little is known about the signal transduction pathways controlling HDAC6 activity. It has been recently described that HDAC6 forms complexes with phosphatases, including the catalytic subunit of PP1, and that HDAC6-PP1 interaction is disrupted by TSA (Brush et al., 2004; Chen et al., 2005). In addition, we have previously reported that tubulin acetylation diminishes in lymphocytes when the activity of RhoA is abolished (Vicente-Manzanares et al., 2002). Furthermore, it is known that Rho GTPases coordinate the two poles of migrating T-lymphocytes (Sanchez-Madrid and del Pozo, 1999; Worthylake and Burridge, 2001) and control the dynamics of actin and tubulin cytoskeleton (Wittmann and Waterman-Storer, 2001; Ridley et al., 2003). It is therefore feasible that HDAC6 has a role in the cross-talk between tubulin- and actin-associated molecules. This role of HDAC6 might explain the relationship between chemokines, Rho/mDia activation/inactivation (Vicente-Manzanares et al., 2002; Palazzo et al., 2004; Destaing et al., 2005) and the consequences of targeting HDAC6 on cell migration (Hubbert et al., 2002; Haggarty et al., 2003; and this report). Whether Rho GTPases could regulate the activity of HDAC6 and its interaction with motor proteins is an interesting point to be explored. We propose that mechanisms such as alteration in signaling or relative PP1, HSP90, mDia, dynein, or tubulin dissociation from molecular complexes with HDAC6 (Kawaguchi et al., 2003; Brush et al., 2004; Bali et al., 2005; Destaing et al., 2005; Hideshima et al., 2005; Kovacs et al., 2005) could modulate lymphocyte migration independently of its deacetylase activity.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
These authors contributed equally to this work.
Address correspondence to: F. Sánchez-Madrid ( fsanchez.hlpr{at}salud.madrid.org)
Abbreviations used: TSA, trichostatin A; NCD, nocodazole; TXL, Taxol; PBL, peripheral blood lymphocyte; FN, fibronectin; MTOC, microtubule-organizing center; Tbc, tubacin; HUVEC, human umbilical vein endothelial cells
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, 1 mM sodium butyrate; 
, 5 µM TSA pretreatment, 1 h.
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