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1 Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
2 Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research (JFCR), 3-10-6, Ariake, Koto-ku, Tokyo 135-8550, Japan
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Abstract |
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Introduction |
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Skeletal muscle differentiation occurs through two steps: generation of skeletal myoblasts and ensuing differentiation of myoblasts into multinucleated myotubes. Appropriate progression of skeletal muscle differentiation requires two groups of regulatory transcription factors, that is, members of the MyoD family and those of the myocyte enhancer factor 2 (MEF2) family. Members of the MyoD family of basic helix-loop-helix (bHLH) transcription factors, including MyoD, Myf5, myogenin and MRF4, are activated by forming heterodimers with ubiquitously expressed E-box proteins, another class of bHLH proteins. These complexes bind to the consensus E-box sequence (CANNTG) present in the promoters of many muscle-specific genes. MyoD and Myf5 are essential for the specification and maintenance of muscle progenitors (Rudnicki et al. 1993), whereas myogenin is required for the terminal differentiation of specified precursors (Hasty et al. 1993; Nabeshima et al. 1993) and MRF4 contributes to the later maturation steps (Olson et al. 1996). Activation of muscle-specific gene expression by the MyoD family is dependent on their association with transcription factors of the MEF2 family. The MEF2 family includes four proteins, that is, MEF2A through D, which bind to conserved A/T-rich sequences in the muscle gene regulatory regions (Gossett et al. 1989). Members of the MEF2 family share two highly conserved domains termed MADS (MCM1, Agamous, Deficiens, serum response factor) and MEF2 domains. The MADS domain mediates homo- and heterodimerization and DNA binding, whereas the MEF2 domain regulates dimerization and co-factor interactions. MEF2 proteins lack myogenic activity themselves, but potentiate the activity of MyoD family factors through combinatorial association and transcriptional cooperation.
Cytokines of the transforming growth factor-ß (TGF-ß) family are multifunctional proteins that regulate the growth, differentiation, apoptosis and morphogenesis of a wide variety of cells (Feng & Derynck 2005). TGF-ß and related factors bind to two different types of serine–threonine kinase receptors, termed type I and type II. Type I receptor is activated by type II receptor upon ligand binding and transduces signals through phosphorylation of receptor-activated Smads (R-Smads). Phosphorylated R-Smads interact with co-Smad (Smad4) and translocate into the nucleus, where Smad complexes regulate transcription of target genes at the Smad-binding sequences in cooperation with transcriptional coactivators or corepressors. Of the eight different mammalian Smad proteins, Smad2 and Smad3 are R-Smads activated by TGF-ß.
TGF-ß influences the differentiation of many types of cells of mesenchymal origin, including myoblasts. TGF-ß inhibits the induction of muscle-specific gene expression and myotube formation independent of cell proliferation (Massague et al. 1986; Olson et al. 1986). Using the TGF-ß type I receptor kinase inhibitor SB431542, we have demonstrated that endogenous TGF-ß signaling suppresses myogenic differentiation of C2C12 cells (Maeda et al. 2004). SB431542 promotes the expression of myosin heavy chain (MHC) and myotube formation in C2C12 cells. Liu et al. reported that Smad3, but not Smad2, acts down-stream of TGF-ß to repress the function of myogenic regulatory factors MyoD and MEF2 (Liu et al. 2001, 2004). They showed that Smad3 physically and functionally interacts with the bHLH domain of MyoD and interferes with the formation of an active MyoD–E-box protein complex and its subsequent binding to multimerized E-box sequences. They also showed that Smad3 interacts with MEF2C through its conserved MADS/MEF2 region and induces displacement of the GRIP-1 coactivator from the MEF2 target promoters.
c-Ski has been shown to act as a negative regulator of TGF-ß family signaling through interaction with co-Smad (Smad4) and R-Smads (Smad2 and 3). The mechanism of Ski-mediated suppression of TGF-ß family signaling is thought to primarily involve transcriptional repression through recruitment of the nuclear corepressor (N-CoR) and histone deacetylases (HDACs) to Smad complexes as well as interference of recruitment of the transcriptional coactivator p300/CBP (Akiyoshi et al. 1999; Wu et al. 2002). Recent structural analysis of the c-Ski–Smad4 complex suggested an alternative model in which c-Ski inactivates the R-Smad–Smad4 complex through inhibition of proper complex formation (Wu et al. 2002), although direct physical interaction between R-Smad and Smad4 is not disrupted by c-Ski (Ueki & Hayman 2003; Takeda et al. 2004). We have recently found that c-Ski enhances binding of Smad complex to DNA and suggested the possibility that inactive Smad complex is stabilized on the promoter region of TGF-ß target genes by c-Ski (Suzuki et al. 2004).
Although many studies have documented the role of c-Ski as a transcriptional repressor, it is still unknown how c-Ski activates the expression of muscle-specific genes. Smad proteins are important partners of c-Ski in mediating its effects, although some activities of c-Ski may occur independently of TGF-ß/Smad signaling. In this study, we investigated the role of c-Ski in muscle differentiation. We found that c-Ski induces myogenic differentiation independently of endogenous TGF-ß/Smad signaling, and through activation of MyoD and inhibition of HDAC activity in the nucleus of myogenic cells.
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Results |
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To determine whether c-Ski-induced myogenin expression is mediated by suppression of TGF-ß signaling, we compared the myogenin promoter activity activated by ectopic c-Ski to that activated by treatment with the low molecular-weight TGF-ß inhibitor SB431542. We generated several myogenin promoter–reporter constructs as shown in Fig. 1A. C2C12 myoblast cells were transfected with each promoter construct and c-Ski, and then incubated in differentiation medium (DM; low-concentration [2%] serum) in the presence or absence of SB431542. c-Ski expression and SB431542 treatment yielded approximately fivefold increase in transcription of the Myo1102-Luc reporter, indicating that elements responsive to both c-Ski and TGF-ß are present from –1102 to –1 in the myogenin promoter. Interestingly, expression of c-Ski induced a threefold increase in the transcription of Myo184-Luc, but treatment with SB431542 failed to activate Myo184-Luc (Fig. 1B). These results suggest that, although both c-Ski and TGF-ß regulate transcription of myogenin, c-Ski acts through the region from –184 to –1 of the myogenin promoter, whereas TGF-ß suppresses transcription through a region up-stream from –184. c-Ski thus activates the myogenin promoter independently of suppression of endogenous TGF-ß signaling.
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Smad3 and Smad4 have been reported to bind to two different DNA motifs, Smad-binding element (SBE; also known as CAGA motif) and TGF-ß inhibitory element (TIE) (Dennler et al. 1998; Yagi et al. 2002; Suzuki et al. 2004). Smad3 binds to DNA upon TGF-ß stimulation, whereas Smad4 binds to DNA independently of TGF-ß (Suzuki et al. 2004). Since c-Ski indirectly binds to DNA through interaction with Smad3 or Smad4, we searched putative Smad-binding DNA motifs from –184 to –1 in the myogenin promoter. Although the TIE motif was not found, one SBE was found at –120 to –117. To examine the importance of the putative SBE in the myogenin promoter, we constructed Myo184mSBE-Luc, in which GTCT was converted to GAGC, and Myo114-Luc, in which the promoter region up-stream from –115 was deleted (Fig. 1A). Although the luciferase activities induced by c-Ski on these mutant reporters were decreased compared to that on Myo184-Luc, significant activation by c-Ski was still observed in both mutant reporters (Fig. 1C). These results suggest that c-Ski-induced activation of Myo184-Luc does not depend on the presence of SBE, and are consistent with the above findings that c-Ski can activate the myogenin promoter independently of regulation of TGF-ß signaling.
c-Ski induces the transcription of myogenin through activation of MyoD, but not that of MEF2C
It has been reported that myogenin expression is regulated by a transcription factor complex containing proteins of the MyoD family and the MEF2 family, whose functions are dependent on the MEF2 binding-site at –67 to –58 in the myogenin promoter. To investigate the effect of c-Ski on the MyoD–MEF2 complex, we first used Myo184mMEF2-Luc, in which the central sequence of the MEF2-site (CTATATTTAT) was replaced by CTCCCGGGAT (Fig. 1A). As shown in Fig. 1C, c-Ski failed to induce transcriptional activation of Myo184mMEF2-Luc, suggesting that c-Ski activates the myogenin promoter through effects on the MyoD–MEF2 complex, and that binding of MEF2 to the myogenin promoter is important for the effects of c-Ski on transcriptional regulation of myogenin.
We next examined the effects of c-Ski on activation of the myogenin promoter induced by MEF2 or MyoD. In C2C12 cells, transfection of either c-Ski or MEF2C induced a fourfold or eightfold increase in promoter activity, respectively. However, transfection of c-Ski together with MEF2C yielded only additive effects (14-fold up-regulation) compared to transfection of c-Ski or MEF2C alone (Fig. 2A, left panel). Furthermore, when the effect of c-Ski on MEF2C was evaluated using a MEF2-responsive reporter (3xMEF2-Luc) in nonmyogenic fibroblast 10T1/2 cells, c-Ski alone neither activated the promoter nor affected MEF2-induced transcriptional activity (Fig. 2A, right panel).
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It has been demonstrated that MyoD is present as an inactive form in muscle precursor cells (myoblasts) (Puri & Sartorelli 2000). To determine whether c-Ski can alter the status of MyoD from inactive to active form, we examined the effect of c-Ski on MyoD activity in growth medium (GM; 10% serum for 10T1/2 cells), in which MyoD is present in the inactive form. As shown in Fig. 2D, MyoD activity in GM was lower than in DM. However, expression of c-Ski was able to up-regulate MyoD activity, suggesting that c-Ski induces activation of MyoD.
c-Ski is unable to augment myogenin expression in the presence of HDAC inhibitors
It has been reported that transcriptional activity of MyoD is suppressed by HDAC1 in myoblasts, and that inhibitors of HDACs promote muscle differentiation and expression of myogenic-specific genes, including myogenin (Iezzi et al. 2002). To examine the effects of HDAC inhibitors on c-Ski-induced MyoD activation, C2C12 cells transfected with c-Ski and MyoD were treated with trichostatin A or sodium butyrate, both of which inhibit class I and II HDACs. Interestingly, although treatment with these inhibitors increased basal activity of the MyoD-responsive reporter 4RE-Luc, c-Ski failed to further up-regulate transcription in the presence of HDAC inhibitors (Fig. 3A). Similar results were obtained with trichostatin A using Myo184-Luc in C2C12 cells (data not shown). Since it has been reported that MyoD does not associate with class II HDACs (Lu et al. 2000), c-Ski may induce MyoD activation through suppression of class I HDACs. We thus examined whether HDAC1-induced suppression of MyoD is released by c-Ski. As shown in Fig. 3B, c-Ski expression overcame MyoD suppression by HDAC1 in GM (20% serum for C2C12 cells). These results suggest that HDAC1 is one of the targets of c-Ski in MyoD activation.
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We further examined the effects of these mutants on myogenin promoter activity using Myo184-Luc and on expression of myogenin and MHC by immunoblotting. Neither c-Ski (ARPG) nor c-Ski (NLSmut.) activated Myo184-Luc (Fig. 4D). Although we detected the expression of myogenin in the absence or presence of the wild-type c-Ski or the NLS. mutant after 66 h in DM, myogenin was detectable at 48 h only in cells expressing wild-type c-Ski, and not in those expressing c-Ski (NLSmut.) (Fig. 4E). Moreover, high expression of MHC was observed at 66 h in DM in the cells expressing wild-type c-Ski, but only weak expression of it was detected in the control and c-Ski (NLSmut.)-expressing cells. These results suggest that localization of c-Ski in the nucleus is essential for induction of muscle differentiation by c-Ski.
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Discussion |
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c-Ski is one of the most potent negative regulators of TGF-ß signaling. Since treatment by a TGF-ß inhibitor was found to promote muscle differentiation (Maeda et al. 2004), it is possible that c-Ski induces muscle differentiation by inhibition of endogenous TGF-ß signaling. Here we demonstrated, using different myogenin promoter constructs, that suppression of myogenin induction by endogenous TGF-ß occurs through a mechanism different from that induced by c-Ski. When we suppressed endogenous TGF-ß with its inhibitor SB431542, promoter activity was increased sixfold in Myo1102-Luc, but was not affected in Myo184-Luc. It has been reported that exogenous TGF-ß signaling regulates MyoD and MEF2, and represses Myo184-Luc promoter activity (Liu et al. 2001, 2004). These findings imply that endogenous TGF-ß signaling regulates myogenin expression through a mechanism different from that by exogenous TGF-ß signaling. The differences in mechanisms of myogenin transcription by endogenous and exogenous TGF-ß signaling might be related to the concentrations of TGF-ß present or amplitudes of TGF-ß signaling. Thus, strong TGF-ß signaling may directly regulate the functions of myogenic regulators MyoD and MEF2, while endogenous weak TGF-ß signaling may control them through yet unknown mechanisms.
We also evaluated whether a Smad-binding element in Myo184-Luc plays roles in c-Ski-induced promoter activation. However, with both point (Myo184mSBE-Luc) and deletion (Myo114) mutants of the myogenin promoters, c-Ski still induced promoter activation, indicating that the effects of c-Ski on muscle differentiation occur independently of TGF-ß signaling.
It has been reported that c-Ski was unable to induce the promoter activity from Myo184 (Ichikawa et al. 1997). In their CAT reporter system, the basal promoter activity of Myo184-CAT was higher than that of Myo1102-CAT, and transfection of c-Ski did not induce up-regulation of Myo184-CAT, suggesting that a suppressor element that may be regulated by c-Ski is present in the region from –1102 to –185. However, the basal activity of Myo1102-Luc was sixfold that of Myo184-Luc in the present study. In addition, we found that a shorter promoter construct, Myo114-Luc, was activated by c-Ski. Further studies are needed to determine whether the region from –1102 to –184 includes additional c-Ski response elements.
In the present study, we demonstrated that c-Ski induces activation of MyoD in C2C12 myoblast cells and nonmyogenic 10T1/2 fibroblasts in both growth and differentiation states. Using HDAC inhibitors, we also showed that MyoD activation may be induced by suppression of HDAC1 by c-Ski. These results are consistent with the finding that c-Ski-induced activation of MCK and MLC1/3 enhancers requires the presence of an E-box (Engert et al. 1995).
Previous studies have suggested a variety of functions for c-Ski in myogenesis. Analysis of c-Ski expression during mouse development showed that up-regulation of c-Ski mRNA in muscle coincided with the formation of secondary myoblasts (Namciu et al. 1995). In c-Ski knockout mice, skeletal muscle mass was reduced, as a result of decrease in fiber size and diameter, but in residual skeletal cells muscle-specific genes, including MyoD, Myf5 and myogenin, are expressed normally (Berk et al. 1997). Since this phenotype is consistent with a defect in expansion or survival of secondary myoblasts, it is clear that c-Ski is essential for neither myogenic determination nor primary myoblast differentiation. This phenotype is similar to that of myogenin-null mice, in which committed myoblasts migrate to their proper locations, although most do not fuse to form myotubes and myofibers, indicating that myogenin is required for secondary myoblast formation but not for commitment to the myogenic lineage (Hasty et al. 1993; Nabeshima et al. 1993).
Mice with a single mutation of either MyoD or Myf5 are viable and do not have an overtly abnormal muscle phenotype, suggesting that MyoD and Myf5 have considerable overlap in their roles in muscle differentiation. However, since double-knockout mice lack essentially all skeletal muscle tissue at birth, either MyoD or Myf5 is required for commitment to the skeletal muscle lineage (Rudnicki et al. 1993). Recently, by evaluating the roles of MyoD and Myf5 using reconstitution assay on MyoD/Myf5 double-knockout fibroblasts, MyoD was found to play a major role in the induction of muscle regulatory factors for terminal differentiation, including myogenin (Ishibashi et al. 2005). Thus, at entry to the process of terminal differentiation, the essential event is myogenin expression, as regulated by activated MyoD and c-Ski might play a role in the activation of MyoD.
In primary undifferentiated myoblasts, MyoD is rendered transcriptionally inactive by several mechanisms. Recently, MyoD has been shown to interact with and be repressed by HDAC1 (Mal et al. 2001; Puri et al. 2001). While the molecular mechanisms leading to HDAC1-mediated inhibition of MyoD remain to be explored, it is likely that the positive function of MyoD induced by acetylation (Sartorelli et al. 1999) may be counteracted by the deacetylase activity of HDAC1. We found that HDAC inhibitors suppressed c-Ski-induced up-regulation of MyoD activity (Fig. 3) and that c-Ski interacts with HDAC1 in C2C12 cells. Thus, HDAC1 may be a target of c-Ski for MyoD activation. On repression of transcription of TGF-ß target genes, c-Ski has been shown to recruit a repressor complex, including HDAC1 (Akiyoshi et al. 1999). Thus, c-Ski has opposing roles in MyoD activation and TGF-ß signaling inhibition. It is currently unknown whether HDAC1 is associated with c-Ski in myogenic cells when c-Ski acts on MyoD, and it will be important to elucidate in the future how c-Ski represses HDAC1 function on the MyoD–HDAC1 complex.
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Experimental procedures |
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C2C12 and C3H10T1/2 cells were obtained from the American Type Culture Collection. C2C12 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 20% fetal bovine serum (FBS) and antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin). The 10T1/2 cells were cultured in DMEM containing 10% FBS and antibiotics.
DNA construction pMyo3700CAT and 3xMEF2-Luc were kindly provided by Dr E.N. Olson (Edmondson et al. 1992; Molkentin et al. 1996). Myogenin promoter luciferase constructs with various lengths and mutations (pMyo1102, 184, 114, mSBE and mMEF2-Luc) were generated from pMyo3700CAT using a PCR-based method and introduced into pGL3-Basic vector (Promega). 4RE-Luc containing four tandem copies of the E-box up-stream of thymidine kinase and luciferase cDNA was previously described (Weintraub et al. 1990). MEF2C was cloned using PCR from a cDNA pool of C2C12 cells and inserted into pcDEF3 expression vector (Goldman et al. 1996). HDAC1, wild-type c-Ski, c-Ski (ARPG) and c-Ski (NLSmut.) were previously described (Akiyoshi et al. 1999; Takeda et al. 2004; Nagata et al. 2006).
Antibodies and chemicals
Preparation of anti-c-Ski antibody was previously described (Nagata et al. 2006). Rabbit polyclonal anti-HDAC1 (H-51) antibodies were purchased from Santa Cruz. Mouse monoclonal anti-myogenin (F5D) antibody was purchased from BD Biosciences, and anti-FLAG (M2) and anti-tubulin antibodies were from Sigma. Anti-MHC antibody (MF-20) was from the hybridoma bank of the University of Iowa. SB431542, trichostatin A and sodium butyrate were purchased from Sigma, and MG132 from Peptide Institute.
Luciferase assay
Cells (1 x 105) were seeded in 12-well plates, followed by transient transfection using FuGENE 6 (Roche Diagnostics) with expression vectors and reporter plasmids. SB431542 (final concentration of 1 µM), trichostatin A (5 nM) and sodium butyrate (5 mM) were added immediately after transfection. At 18 h after transfection, cells were transferred to DM (DMEM containing 2% FBS) and then incubated for 24 h. Luciferase activity in cell lysates was determined by a dual luciferase reporter assay system (Promega) using a luminometer (AutoLumat LB953, EG & G Berthold) and normalized to sea-pansy luciferase activity of co-transfected phRL-TK (Promega).
Fractionation of nuclear and cytoplasmic proteins
Fractionation of nuclear and cytoplasmic proteins was performed as described (Andrews & Faller 1991). Briefly, 1 x 105 cells were transfected with expression vectors (total amount of 1 µg) using FuGENE 6. After 48 h, cells were washed with PBS and collected in Buffer A (10 mM HEPES–KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and 50 U/mL aprotinin) and incubated on ice for 10 min. After centrifugation, the supernatant was used as a cytoplasmic fraction. The precipitate was washed with Buffer A and suspended in Buffer C (20 mM HEPES–KOH, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and 50 U/mL aprotinin). The suspension was incubated on ice for 20 min and the supernatant obtained by centrifugation was used as a nuclear fraction. Proteins of each fraction were mixed with Laemmli sample buffer (50 mM Tris–HCl, pH 7.0, 4% SDS, 10% 2-mercaptoethanol, 1 mM EDTA, 10% glycerol and 0.01% bromophenol blue) and analysed by SDS-PAGE and immunoblotting. HRP-conjugated M2 antibody (Sigma) was used for detection of c-Ski and its mutants, and reacted antibodies were detected with immunofluorescence using the ECL system (Amersham, Piscataway, NJ).
Myogenic differentiation
Muscle differentiation was determined by immunohistochemical staining to count multinuclear cells and immunoblotting for determination of the expression of myogenin and MHC proteins. For immunohistochemical analysis, C2C12 cells (1.5 x 105) were seeded in 6-well collagen I-coated plates (Sumitomo). At 18 h after transient transfection, cells were transferred to DM and further incubated for 5 days. Cells were fixed in 1 : 1 acetone–methanol solution for 5 min, washed with PBS and blocked with Blocking One (Nacalai Tesque, Kyoto) for 15 min. Wild-type c-Ski and mutants of c-Ski were detected by incubation with anti-c-Ski antibody for 1 h. Cells were washed with PBS and subsequently stained with secondary antibodies conjugated with Alexa Fluor® 488 anti-rabbit IgG antibody (Invitrogen Molecular Probes). The nuclei were stained by propidium iodide (Invitrogen Molecular Probes). Fluorescence was examined using an Olympus IX71 microscope. To determine the expression of myogenin and MHC proteins, 5 x 104 cells were seeded in 6-well collagen IV-coated plates (IWAKI). At 24 h after transient transfection, cells were transferred to DM and further incubated for 48 and 66 h. Cells were harvested with lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 50 U/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride) and protein concentrations were quantified by the Bradford method, and extracts containing 30 µg of total proteins were analysed by SDS-PAGE and immunoblotting.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: miyazono-ind{at}umin.ac.jp
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References |
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Received: 14 August 2006
Accepted: 11 December 2006
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-tubulin antibodies were used as nuclear (N) and cytoplasmic (C) markers, respectively. c-Ski from the nuclear fraction migrated more slowly than that from the cytoplasmic fraction, possibly due to the difference in the buffers used for solubilization and/or post-transcriptional modification of the protein.
(D) C2C12 cells were transfected with Myo184-Luc and the indicated c-Ski plasmids, and luciferase activity was measured as described in Experimental procedures.
(E) C2C12 cells were seeded on collagen IV-coated 6-well plates and transiently transfected with mock (C), FLAG-tagged c-Ski wild-type (W), or NLS mutant (N) plasmids. At 24 h after transfection, cells were transferred to DM and further incubated for 48 or 66 h. Expression of c-Ski proteins was detected with anti-FLAG antibody (upper panel), and expression of myogenic protein markers was detected with anti-myogenin and anti-MHC antibodies. Anti-