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SOLUBLE FACTOR PRODUCED BY MYOTONIC DYSTROPHY MYOBLASTS INHIBITS MYOGENIC DIFFERENTIATION.

Daniel Beaulieu 1 , Caroline Haineault 1 , Gilles Doucet 1 , Denis Furling 2 and Jack Puymirat 1 *

1Laboratory of Human Genetics, CHU Laval Research Center, Quebec, Canada, G1V 4G2;
2CNRS UMR 7000, Hôpital Pitie-Salpetriere, Paris, France.

* Address of Correspondence to:

Jack Puymirat,
Laboratory of Human Genetics,
Laval University Medical Research Center.
CHUQ Pavillon CHUL.
2705 Laurier Blvd, Sainte-Foy, Quebec, Canada, G1V 4G2.
Tel: (418) 654-2186, Fax: (418) 654-2207. E-mail: Jack.Puymirat@crchul.ulaval.ca

La dystrophie myotonique de type 1 (DM1), la maladie neuromusculaire la plus fréquente chez l’adulte, est causée par une répétition de CTG localisée sur le chromosome 19q23. Un retard de développement du système musculaire est observé chez les patients atteints de la forme congénitale de la maladie, tandis que dans la forme adulte de la maladie, la régénération des fibres musculaires est déficiente. Les mécanismes moléculaires par lesquels l’expansion de CTG affecte le développement ou la régénération du système musculaire sont inconnus.

Nous avons montré, in vitro , que l’inhibition de la différenciation myogénique observée chez les myoblastes prélevés de patients atteints de DM1 est proportionnelle à la longueur de l’expansion des CTG. Ce défaut est associé à une diminution spécifique de l’expression de la myogenin. De plus, nous avons démontré qu’un facteur soluble de masse moléculaire inférieure à 30 kDa est sécrété par les myoblastes DM1 et inhibe la différenciation terminale des cellules musculaires précurseurs. L’effet de ce facteur soluble est associé à une diminution spécifique de l’expression de la myogenin et est proportionnel à la longueur de l’expansion des CTG. Nos résultats suggèrent que la mutation caractéristique de la DM1 induit l’expression d’un facteur soluble inhibant la différenciation terminale des cellules musculaires précurseurs. Ces résultats devraient avoir un impact majeur sur la compréhension des mécanismes moléculaires responsables des défauts de maturation et de régénérescence du système musculaire observés dans la forme congénitale et adulte de la DM1.

Myotonic dystrophy (DM1), the most common form of inherited neuromuscular disease, is caused by a CTG repeat expansion at chromosome 19q23. Fetal muscle development is affected in patients with a congenital form of the disease, and abnormalities in muscle regeneration have been reported in patients with adult onset of the disease. The mechanisms by which the DM1 mutation affects skeletal muscles development or regeneration are unknown. In primary human DM1 satellites cell cultures, we showed that the defect of DM1 myoblasts are deficient in their ability to fuse and that this defect was proportional to the length of the CTG repeat tract. In addition, we showed that their impairment to fuse was associated with a specific reduction in myogenin gene expression. We have identified a soluble factor with a Mr < 30-KDa that is secreted by DM1 myoblasts and which blocks myogenic differentiation. This inhibitory effect was associated with a specific decrease in myogenin gene expression and was directly proportional to the length of the CTG repeat expansion. Our results suggest that the DM1 mutation triggers the expression of a soluble factor in DM1 myoblasts which inhibits muscle cell differentiation. This could have a major impact in understanding the delay in muscle development observed in the congenital form of the disease or the defects in muscle regeneration in adult patients.

Key words: Myotonic dystrophy-myoblasts-differentiation-myogenin-soluble factor.

Myotonic dystrophy (DM1), the most common form of inherited neuromuscular disease in adults, affects 1 in 8000 individuals worldwide. DM1 is an autosomal dominant muscular dystrophy with very variable symptom presentations. Adult onset DM1 is characterized by myotonia, muscle wasting and weakness, cataracts, cardiac conduction abnormalities, testicular atrophy and insulin resistance (1). Moreover, the disease is associated with specific defects in muscle differentiation. Abnormal muscle regeneration has been reported in patients with adult onset DM1 (2), and alteration in muscle development has been described in the severe congenital form of the disease (CDM1) (3,4).

DM1 is caused by the expansion of an unstable CTG trinucleotide repeat in the 3’untranslated region of the myotonic dystrophy protein kinase (DMPK) gene (5-7). The physiopathological mechanisms responsible for the varied symptoms of DM1 are still not thoroughly understood. Two mechanisms have been proposed to explain how the CTG amplification causes the complex, multisystemic DM1 phenotype. First, the expansion affects the levels of DMPK expression or that of neighboring genes leading to haplo-insufficiency (8-12). Second, mutant DMPK transcripts carrying an expanded CUG repeat are sequestrated in the cell nucleus, forming nuclear foci, and appear to have a detrimental effect on the metabolism of other mRNAs ( trans- effect) leading to myotonia and probably to the majority of the symptoms (13-19, reviewed in ref 20). To date, mouse models have not reproduced the muscle development defects seen in DM1. Neither the loss-of-function nor overexpression of DMPK in mice has resulted in muscle development abnormalities (21, 22). Recent experiments carried out in transgenic mice expressing an untranslated expanded CUG repeat under the control of the human skeletal actin promoter showed that expanded CUG repeats are sufficient to generate DM1-like myopathy (23). However, skeletal muscle development defects were not observed in these mice. More recently, it was shown that the mouse knockout of the muscleblind gene, whose product is trapped in the DM1 nuclear inclusions, reproduces the phenotype of the disease but skeletal muscle development defect was not observed in these animals (24). While CDM1 and adult onset forms of the disease share the characteristic trinucleotide amplification, the molecular mechanisms leading to delay in muscle maturation in the CDM1 form are still poorly understood. Growth of skeletal muscle is mediated by satellite cells that proliferate and fuse with growing fibers. In adult muscle, satellite cells coordinate muscle regeneration to repair muscle damage. Myogenesis is governed by the basic helix-loop-helix myogenic regulatory transcription factors (MRFs) MyoD, Myf5, MRF4 and myogenin (reviewed in ref. 25). Genetic studies in mice have shown that MyoD, Myf5 and MRF4 are involved in the commitment of cells to the myogenic lineage (i.e., becoming myoblasts), whereas myogenin is required for myoblast differentiation (25, 26). Controversial data have been reported on the levels of MRFs in DM1 skeletal muscle. Using the C2C12 myoblasts model system, MyoD has been identified as a target of mutant DMPK 3’UTR RNA, (27, 28). On the opposite, it was reported that overexpression of CUG repeats within the mutant DMPK mRNA inhibit terminal differentiation of murine myoblast cell line C2C12 and that this effect was associated with a specific decrease in myogenin gene expression (29). Similarly, it was also shown that skeletal muscle cells from DM1 patients fail to induce cytoplasmic levels of a CUG RNA binding protein, CUGBP1, which leads to a significant reduction of p21 gene expression and to alterations of other proteins involved in cell cycle withdrawal (30).

In this paper, we identified a soluble factor produced by DM1 myoblasts that blocks myogenic differentiation. The inhibitory effect was proportional to both the deficiency of DM1 myoblasts to fuse and to the length of the CTG repeat track. In addition, the delay in muscle differentiation was associated with a specific decline in myogenin gene expression. We hypothesized that the DM1 mutation triggers the production of a soluble factor, in DM1 myoblasts, which blocks their differentiation.

Primary human myoblast culture :

Normal human myoblasts were derived from a 15-week-old fetus (obtained from Clonetics, USA) and from a neonate infant who died during the neonatal period. DM1-750, DM1-1200 and DM1-3200 myoblasts were obtained from the skeletal muscles of 20-, 13- and 15-week-old DM1 fetuses. The length of the CTG repeat tract in these myoblasts was approximately 750, 1200 and 3200 CTG repeats, as determined by Southern blot. Because these myoblasts were obtained from aborted fetuses, we did not determine if they had DM1 or CDM1. Two other DM1 myoblasts cell types (CDM1-3300 and CDM1-3700) were derived from two 28 and 34-week old fetuses that were diagnosed as CMD1. The length of the CTG expansion was approximately 3300 and 3700 CTG repeats in these two CDM1 myoblast cell types. All biopsies were obtained in accordance with the Laval University Medical Research Centre ethical committees.

Myoblasts were grown in MCDB 120 medium supplemented with 15% heat-inactivated fetal bovine serum, 5 μ g/ml insulin, 0.5 mg/ml BSA, 10 ng/ml epidermal growth factor, 0.39 μ g/ml dexamethasone, 50 μ g/ml streptomycin and 50 μ g/ml penicillin (proliferative medium), as previously described (31). For myoblast differentiation, the cells were subsequently cultured in DMEM supplemented with 0.5% heat-inactivated fetal bovine serum, 10 μ g/ml insulin, 10 μ g/ml apo-transferrin, 50 μ g/ml streptomycin and 50 μ g/ml penicillin (differentiating medium). All cultures were incubated at 37ºC in a humid atmosphere containing 5% CO2. Normal and DM cells were used between the 4th and 6th passage. The number of passages refers to the total number of passages from the time following the isolation of the initial myoblast population from the fetuses.

Preparation of conditioned medium:

Conditioned medium (CM) was prepared from normal myoblasts, DM1 myoblasts with 750, 1200, 3200, and from two CDM1 myoblasts with 3300 and 3700 repeats. At 70% of confluence, the cells were washed with HBSS for one hour to remove residual serum and were then grown in proliferative medium containing 15% FBS for 2 days (MCDB120-15%FBS CM). The medium was then collected and frozen until later use. The cells were then washed with HBSS and were further propagated in serum-free proliferative medium (MCBD120-serum free CM) for 2 days. The medium was then collected and frozen until later use. MCDB120-15%FBS CM was added to normal myoblasts at 70% confluence. Two days later (90% confluence), the cells were switched to MCDB120-serum-free CM and were further grown in this medium for the next four days. CM was renewed every day. Non-conditioned with serum and serum-free proliferative media were used as controls.

Fractioning of conditioned medium:

Conditioned medium was generated using DM1 myoblasts with 3200 repeats or normal myoblasts as described above. Conditioned media prepared with or without serum were fractionated on Amicon Ultra-15 Centrifugal Devices with a 30 kDa of nominal molecular weight limit (Millipore, USA). Non-conditionated proliferative and differentiating medium were also fractionated in a similar way. CM containing proteins with a MW below 30-kDa was supplemented with fractionated non-conditionated medium containing proteins with a MW above 30-kDa, in order to replenish the components in the medium that are necessary for cell proliferation, and which may have otherwhise been depleted in the fractionation procedure. CM containing proteins with a MW above 30-kDa was supplemented with fractionated non-conditionated medium containing proteins with a MW below 30-kDa. The fractioned CM with serum was added to normal myoblasts when they reached 70% confluence. At confluence, the serum-free fractioned medium was added to the cells to induce differentiation. The fusion index was determined after 4 days of differentiation by counting the number of nuclei in multinucleated cells and was expressed as a percentage of the total number of nuclei.

Western blot analysis

The cells were scraped off after 4 days of differentiation. The complete procedure for the extraction of cytoplasmic and nuclear proteins used is described by Jansen et al. (21). Electrophoresis samples were solubilized in Laemmli’s buffer and boiled for 5 min. Proteins (20 μ g) were separated by SDS-PAGE on a 10% polyacrylamide resolving gel slab and were transferred to PVDF membranes (BioTrace, Pall Coproration) by electro-blotting. After blocking non-specific sites, the membranes were incubated with primary antibodies: anti-myogenin (Clone F5D, DSHB, Iowa), anti-myoD (BD PharMingen) or anti-p21 (Bio/Can Scientific). The membranes were washed, and incubated with an anti-immunoglobulin secondary antibody conjugated to horseradish peroxidase. The immune complexes were detected using an enhanced chemioluminescence kit (ECL, Amersham).

Southern blot:

Genomic DNA was extracted by using proteinase K combined with the denaturing ability of the ionic detergent SDS, as previously described (32). Twelve μ g of genomic DNA were used for each digestion with appropriate restriction enzymes, and the samples were purified by phenol-chloroform extraction prior to gel loading. The DNA was resolved on an 0.8% agarose gel, blotted onto a Biodyne B nylon membrane (BioTrace, Pall Corporation) and hybridized to a 32P-labelled DMPK probe according to standard procedures.

Differentiation of DM1 myoblasts

DM1, CDM1 and normal muscle cells used in this study were purified by cell sorting (FACS) using a neural cell adhesion molecule (N-CAM) antibody, as previously described (31). More than 90% of DM1 muscle cells were vimentin- and desmin-positive indicating that they are myoblasts (data not shown). When normal human myoblast cultures were switched from permissive to non-permissive conditions, the majority of the cells ceased DNA synthesis by 48 h, myotubes formation began within 3 days in non permissive conditions, and myotubes continued to increase in size and number over several days. The fusion index of control cultures was about 80% after 4 days in differentiation medium. As seen in Figure 1B, the fusion index of DM1 myoblasts was inversely proportional to the length of the CTG repeat tract (65%, 55% and between 10 to 30% for DM1 myoblasts with 750, 1200 and between 3200 to 3700 CTG repeats, respectively). Although the proliferating DM1 and normal myoblasts have very similar morphology in growth medium, the myotubes formed by both types of cells showed striking differences. After 4 days in differentiating medium, the normal myoblast cultures (control) formed large myotubes with an average of 13 nuclei per cell (fig.1A) whereas DM1 myotubes were much smaller, thinner and had a relatively small number of nuclei per myotube. DM1 with 750 repeats contained an average of 8 nuclei per myotube, DM1 with 1200 repeats, 7 nuclei per myotube, and DM1 with 3200 to 3700 CTG repeats an average of 3 nuclei per myotube .

The clinical phenotype for DM1-750, DM1-1200 and DM1-3200 myoblasts was unconfirmed because of the early age of the fetuses (20, 13 and 15 weeks respectively). The only reliable feature to diagnose the congenital form of the disease, at the molecular level, is the examination of the proximal region of the CTG expansion known to be aberrantly methylated in CDM1 (33). We have therefore set out to determine the methylation status of the normal, DM1-750, DM1-1200, DM1-3200 DM1 myoblasts, as well as for the two CDM1 cells used in our assays. DNA prepared from the various myoblasts was digested using methylation-sensitive Sac- I and Sac -II restriction enzymes and analyzed by Southern blot, as described (33). Normal DMPK alleles generate a single 1.8 Kb fragment following co-digestion by Sac -I and Hind -III, whereas mutant alleles generate a band augmented by the corresponding length of the CTG expansion (Fig. 2). While the upstream Sac -II site is constitutively methylated in normal, DM1-750 and DM1-1200 cells, the Sac -II site proximal to the repeat tract is aberrantly methylated in the two CDM1 cell lines and in DM1-3200. Triple digestion with Sac- I, Sac -II and Hind -III results in the excision of the repeat tract in normal and DM1-750 and -1200 myoblasts, whereas the expanded tract was retained in DM1 myoblasts with 3200, 3300 and 3700 repeats, as revealed by 11.4-, 11,7- and 12,9- kb DNA fragments. These restriction assays demonstrate that methylation occurs only in the mutant DMPK gene of DM1-3200, CDM1-3300 and CDM1-3700 cells. This confirms that methylation occurs in CDM1 myoblasts. The fact that methylation also occurred in the mutant allele of DM1-3200 strongly suggests that these myoblasts were derived from a CDM1 fetus. This possibility is also supported by the fact that the mutated allele was transmitted by the mother of the fetus from which DM1 myoblasts with 3200 repeats were derived. It is indeed well established that the congenital forms of the disease are only transmitted by the mother. In addition, our data also indicate that DM1-750 and DM1-1200 myoblasts are most likely not derived from CDM1 fetuses. This is also supported with the observation that the mutation was transmitted by the father of the two fetuses from which these two DM1 myoblasts were generated.

Effect of conditioned medium on index of fusion

Conditioned media (CM) were prepared as described in materials and methods. When normal myoblasts were grown in the medium used for CM but which has not been incubated with cells, there was a significant decrease in the index of fusion of control myoblasts (from 80% for control myoblasts to 40-50% for cells grown in the medium used for CM). This decrease may be explained by the presence of proliferative components in the medium used during differentiation. CM generated from normal myoblasts had no significant effect on the fusion index of control myoblasts. In contrast, CM generated from DM1 myoblasts inhibited the fusion of normal myoblasts (Fig. 3) and this effect was proportional to the length of the CTG repeat tract in the DM1 myoblasts that have been used to prepare the CM.

Conditioned medium blocks Myogenin Gene Expression

The impairment of DM1 myoblasts to fuse suggests an alteration in the expression of MRFs. Based on previously published data, we examined the expression of myogenin and MyoD in normal and DM1 myoblasts (Fig. 4). The expression of myogenin, a muscle-transcription factor activated specifically in differentiated muscle cells, was significantly reduced in DM1 myoblasts and this decrease directly correlated with extent of the expansion (Fig. 4B). In contrast, the expression of MyoD was not significantly altered (Fig. 4A). To determine whether CM also alters myogenin gene expression, we studied the effects of CM on myogenin and MyoD gene expression in normal myoblasts incubated with CM. As shown in Fig 4, CM generated from DM1 myoblasts specifically induced a reduction in myogenin gene expression whereas no significant effect was observed on the levels of MyoD. This decrease was also directly correlated with the length of the CTG repeat tract. In addition we verified the expression of p21, whose reduced levels were previously reported in DM1 muscle cells. As shown in figure 4C, p21 gene expression remained unchanged in DM1 myoblasts whatever the length of the expansion, as well as in control myoblasts incubated with CM generated from DM1 cells.

A Soluble Factor Inhibits The Differentiation of Normal Myoblast s

As a first attempt to identify this factor, we have fractionated CM in more manageable fractions containing proteins with either smaller than 30 kDa or larger than 30 kDa. We then evaluated the ability of these individual fractions to block the differentiation of normal myoblasts (Fig 5). The differentiation of normal myoblasts grown in CM in the presence of proteins with a MW above 30 kDa was very similar to control myoblasts, whereas the differentiation of myoblasts incubated in the presence of proteins with a MW below 30 kDa blocked their fusion to a level similar to the non fractionated CM (Fig 5A). The fusion index of normal myoblasts incubated with unfractionnated CM and CM containing proteins with a Mr ‹ 30 kDa was 5% and 6%, respectively (Fig. 5B). In contrast, the fusion index for myoblasts incubated with CM containing protein with a Mr › 30 kDa was similar to the control (39% and 42%, respectively). These data indicate that a soluble factor that blocks myogenic differentiation has a Mr smaller than 30 kDa.

A major defect observed with myoblasts isolated from DM1 patients is a deficient in their ability to fuse. This defect is proportional to the length of the CTG repeat tract. This impairment to fuse suggests an alteration in the expression of MRFs (25). Here we showed that the fusion defect of DM1 myoblasts is associated with a specific reduction in myogenin gene expression, whereas myoD gene expression was not altered. Because DM1 myoblasts are all desmin-positive, this indicates that the differentiation of satellite cells into myoblasts following activation, a myogenic pathway under the control of MyoD, is not altered by the DM1 mutation. Overall, our data agree with genetic studies in mice showing that myogenin is required for myoblast differentiation (reviewed in ref 25). In addition, we showed that the levels of p21 protein remain unaffected in DM1 myoblast cultures, even when they have lost their ability to fuse. This implies that p21 is most likely not involved in the impairment of DM1 myoblasts to differentiate as previously suggested (30). Together our data suggests that the fusion defect of DM1 myoblasts to fuse most likely results from a specific reduction in myogenin gene expression.

In this study, we have identified a soluble factor produced by DM1 myoblasts that blocks myogenic differentiation. The observation that this factor produces concomitant effects on MRFs gene expression, as we observed in DM1 myoblasts strongly suggests that it may be involved in their deficiency to fuse. We also showed that the ability of CM to inhibit myoblast differentiation is proportional to the length of the CTG repeat tract of DM1 cells used to generate the conditioned medium. This suggests that the production of the soluble factor is determined by the extent of the CTG repeat tract. This also indicates that this soluble factor is not solely produced by CDM1 cells, and that other factors must be involved in the muscle atrophy and the delay in maturation observed in CDM1 cells. A role for a maternal environmental factor in the pathogenesis of DM1 has been postulated because CDM1 only occurs in the offspring of DM1 mothers (1). A factor from maternal serum has previously been identified, based on data showing that rats injected with this human serum show a delay in muscle maturation (3). Whether the factor identified in the present study is the same as the one identified in above study remains to be determined. In the affirmative, this would indicate that this protein is not produced by the mother, as previously suggested, but by the skeletal muscles of DM1 fetuses.

The identification of a soluble factor produced by DM1 satellite cells which may contribute to the delay in myoblast differentiation for the congenital form of the disease and in the deficiency of muscle regeneration in the adult form, opens a new avenue in our understanding of the pathogenesis of muscle weakness and wasting in DM1. The identification of this soluble factor is presently under investigations.

The authors would like to thank Dr Marc-André Langlois for his technical assistance. This research was funded by grants to J.P. from the MDA (USA), the French Myopathy Association (AFM), The Institutes of Health Research of Canada (IRSC) and the Canadian Association for Muscular Dystrophy (ACDM). We thank Dr J. David Brooke for the pM10M6 DMPK plasmid.

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Footnotes

1 Abbreviations used are: DM1 myotonic dystrophy type 1; CDM1, congenital myotonic dystrophy type 1; DMPK, myotonic dystrophy protein kinase; CM, conditioned medium.

Figure 1. Index of fusion of DM1 myoblasts. ( A ) Normal myoblasts (Normal), DM1 myoblasts with 750 (DM1-750), 1200 (DM1-1200) and 3200 (DM1-3200) repeats and CDM1 myoblasts with 3300 (CDM1-3300) and 3700 (CDM1-3700) repeats were fixed and stained with DAPI after 4 days of differentiation. ( B ) Quantitation of the fusion index for DM1 myoblasts with different expansion sizes. The number of nuclei in multinucleated myotubes was expressed as a percentage of the total number of nuclei. About 1000 nuclei from three independent experiments were counted. (* P < 0.05, ** P < 0.01 *** P < 0.001 ; t’test)

Figure 2 . Southern blot. Analysis of genomic DNAs from normal ( A ), DM1-750 ( B ), DM1-1200 ( C ), DM1-3200 ( D ), CDM1-3300 ( E ) and CDM1-3700 ( F ) cells. Restriction enzymes used for genomic digestions are the following : Sac- I, Sac -II and Hind III. The asterisk (*) indicates the DNA fragment containing the aberrantly methylated downstream Sac -II site in DM1-3200, CDM1-3300 and CDM1-3700 myoblasts. Bands at 4.0-, 5.4-, 11.4 -, 11,7- and 12,9-kb represent full-length ( Sac -I to Hind III) mutant allele of DM1-750, DM1-1200, DM1-3200, CDM1-3300 and CDM1-3700 myoblasts, respectively. Bands at 1,8 kb represent the full-length normal allele in all myoblasts.

Figure 3. Alteration in the fusion index of normal Myoblasts exposed to the conditioned medium. ( A ) Control myoblasts were incubated with conditioned medium (CM) from normal (N-CM), DM1 with 750 (750-CM), 1200 (1200-CM) and 3200 CTG (3200-CM), CDM1 with 3300 (CM-3300) and 3700 (CM-3700 repeats. After 4 days of differentiation, cells were fixed with formaldehyde and stained with DAPI. ( B ) Quantitation of the fusion index. The number of nuclei in multinucleated myotubes was expressed as a percentage of the total number of nuclei. About 1000 nuclei from three independent experiments were counted. (* P < 0.05, ** P < 0.01 and *** P < 0.001; t’test)

Figure 4. MyoD, myogenin and p21 protein expression. Left panel : Protein levels for MyoD ( A ), myogenin ( B ) and p21 ( C ) in normal and DM1 myoblasts with 750, 1200 and 3200 CTG repeats, as determined by Western blotting. Right panel : Protein levels in normal myoblasts grown in the presence of CM generated from DM1 myoblasts with 750, 1200 and 3200 repeats. ( D ) A characteristic Western blot with nuclear-cells extracts prepared from differentiated normal (left panel) and DM1 (right panel) myoblasts grown in CM and probed with the indicated antibodies. (* P < 0.01, t’test, NS : insignificant data)

Figure 5. Effect of fractionated conditioned medium on the index of fusion . Normal myoblasts were either grown in unfractionated CM or fractionated conditioned medium (CM < 30 kDa and CM > 30 kDa), both generated from DM1 myoblasts with 3200 repeats after 4 days in differentiating medium. ( A ) Cells were stained with DAPI. ( B ) Fusion index of normal myoblasts incubated with conditioned medium containing proteins larger than 30 kDa (> 30 kDa) or smaller than 30 kDa (< 30 kDa). One thousand (1000) nuclei were counted (***P < 0.001; t’test)

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© Daniel Beaulieu, 2005