RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and purified as previously described (Ozawa and Kobayashi, 2014). surface localization of endogenous M-cadherin and N-cadherin, as well as cellCcell fusion. This domain, however, does not inhibit myoblast differentiation according to microarray-based gene expression analysis. In contrast, expressing a dominant-negative -catenin mutant ectopically, which suppresses Wnt/-catenin signaling, did not inhibit cellCcell fusion. Therefore, the E-cadherin cytoplasmic domain inhibits cellCcell fusion by inhibiting cell surface localization of endogenous cadherins and not by inhibiting Wnt/-catenin signaling. (Hollnagel et al., 2002). The presence of multiple cadherins in myoblasts has hindered the study of their individual Olprinone Hydrochloride roles in skeletal muscle fusion and are already expressed before differentiation is induced, and myogenin transcription is upregulated upon myogenic induction (Olson and Klein, 1994). Consistent with these observations, microarray analysis of C2C12 cells expressing either DsRed or DECT revealed that and mRNA are already present before differentiation is induced and did not change in level following induction (Table?1). Myogenin mRNA expression was upregulated upon myogenic induction in DsRed+ and DECT+ C2C12 cells (Table?1). As myogenin activity is crucial for activating the entire differentiation program, we determined its protein level using immunoblotting. The expression of myogenin protein (MyoG) was elevated in cultures of both DsRed+ and DECT+ cells, even though the latter rarely fuse into myotubes (Fig.?3). Therefore, DECT expression did not affect the upregulation of myogenin expression following myogenic differentiation. As shown in Table?1, the expression of other differentiation-regulated genes was either upregulated or downregulated upon induction of differentiation in DsRed+ and DECT+ cells. Table?1. Changes Olprinone Hydrochloride in relative expression levels of marker genes in C2C12 cells expressing either DsRed or DECT upon myogenic differentiation Open in a separate window Open in a separate window Fig. 3. Immunoblot analysis of differentiation-associated markers. Cells cultured in differentiation medium for 5?days were subjected to immunoblot analysis with the antibodies indicated on the panel. Vinculin was used as a loading control. Myogenin protein levels were upregulated in both DsRed+ and DECT+ cells. -catenin signaling activation during myogenic differentiation The Wnt/-catenin signaling pathway has been reported to play key roles in myogenic fate determination and differentiation (Cossu and Borello, 1999; Petropoulos and Skerjanc, 2002; Ridgeway et al., 2000). Canonical Wnt signaling is also reportedly involved in myogenic differentiation of mouse myoblasts (Tanaka et al., 2011). Since -catenin is a critical player in the Wnt signaling pathway (Clevers, 2006), -catenin sequestration by cadherin’s cytoplasmic domain has been shown to block its nuclear translocation and therefore inhibit -catenin-mediated transcription activity (Sadot et al., 1998; Orsulic et al., 1999; Simcha et al., 2001). Inhibition of -catenin signaling by DECT may result in the suppression of myogenic cell differentiation and myotube formation. To determine whether -catenin signaling is activated during C2C12 cell differentiation, we performed a reporter assay using a transgenic construct that expresses EGFP under the control of tandem repeats of a LEF-1/TCF binding site (TOP-EGFP) (Korinek et al., 1997) (Fig.?4A). The same construct was previously used to successfully monitor LEF-1Cdependent -catenin activity (Arnold et al., 2000). The construct was introduced into C2C12 cells and Olprinone Hydrochloride stable transfectants were isolated. When TOP-EGFP+ cells were cultured in growth medium, they exhibited low levels of EGFP protein as determined by immunofluorescence staining (Fig.?4B). When the cells were induced KSHV K8 alpha antibody to differentiate under low serum conditions, they expressed high levels of EGFP (Fig.?4C). Although high levels of EGFP were detected in giant multinucleated cells, the area showing a strong EGFP signal did not coincide with the area showing strong MHC staining signal (Fig.?4C), raising the possibility that the Wnt/-catenin signaling pathway becomes inactivated either during the later stages of differentiation or after myoblast fusion. In any case, activation of the Wnt/-catenin signaling pathway occurs during the differentiation of C2C12 cells. Open in a separate window Fig. 4. The Wnt/-catenin pathway is activated during C2C12 cell differentiation. (A) Schematic representation of the Wnt/-catenin pathway reporter construct, TOP-EGFP. The red boxes indicate three LEF-1/TCF-binding sites in the promoter region followed by a green box representing the EGFP coding sequence. (B) A stable C2C12 transfectant harboring TOP-EGFP cultured in.