Stx4 is expressed in skeletal muscles and enhanced during myoblast differentiation

In the previous study, we performed a yeast two-hybrid screening to identify interacting proteins for Cdo, and JLP and Bnip2 are two such proteins implicated in Cdo-mediated myogenesis [13, 14]. In the same screen, Syntaxin (Stx) 1 was identified as an interacting protein for Cdo. Stx1 and Stx4 share high homology and have similar domain structures consisting of Stx, t-SNARE, and transmembrane domain (TD) [28]. While Stx1 is expressed predominantly in neural cell types, Stx4 is the major form in skeletal muscles [29, 30]. Therefore, we examined whether Stx4 plays a role in myogenesis, especially in association with Cdo. First, we have assessed the expression pattern of Stx4 and Cdo in mouse hindlimb muscles from various developmental stages. The expression of Stx4 was detected throughout the examined stages; however, the level of Cdo, MyoD, and Myogenin decreased after the postnatal day 7 which may reflect the fast muscle growth during early postnatal life (Fig. 1a). Next, we have examined the expression pattern of Stx4 protein during myoblast differentiation. C2C12 cells were grown to near-confluency (D0) and induced to differentiate by switching to the differentiation medium for a total of 3 days (D3), followed by immunoblotting. As shown in Fig. 1b, the level of Stx4 is enhanced progressively during myoblast differentiation, while the Cdo protein is expressed throughout the differentiation time course, and the expression of Myogenin and myosin heavy chain (MHC) was dramatically enhanced at D2 or D3, respectively. These data suggest that Stx4 might be important for myoblast differentiation. Since Cdo and Stx4 were coexpressed in developing skeletal muscles, we examined the relationship between Cdo and Stx4 by using primary myoblasts isolated from Cdo +/+ or Cdo −/− mice. Previously, we have shown that Cdo-deficient primary myoblasts display defects in myoblast differentiation and p38MAPK activation [26]. Cdo +/+ or Cdo −/− myoblasts at high cell density (D0) were induced to differentiate by removal of basic fibroblast growth factor (bFGF) for 2 days. The expression of Stx4 in Cdo −/− myoblasts was substantially increased at D2 compared to that of Cdo +/+ myoblasts, whereas there was only slight or no difference at D0 and D1 (Fig. 1c). In addition, the qRT-PCR analysis showed that Stx4 transcript levels were increased at D1 in Cdo-deficient myoblasts, but no difference in cells at D0 or D2 (Fig. 1d). These data suggest that the Stx4 expression level alone may not be sufficient to induce myoblast differentiation when Cdo is deficient.

Fig. 1 Stx4 is expressed in skeletal muscles and induced in myoblast differentiation. a RT-PCR analysis of hindlimb muscles from E15.5 embryos and P1, P5, P7, P14, and P30 mice for the expression of Stx4, Cdo, MyoD, Myogenin, and 18S rRNA serves as a loading control. b Immunoblot analysis of C2C12 cells from various differentiation days (D) for the expression of Stx4, Cdo, Myogenin, MHC, and pan-Cadherin serves as a loading control. c Immunoblot analysis for Stx4 protein expression in Cdo +/+ and Cdo −/− primary myoblasts during differentiation, and pan-Cadherin serves as a loading control. d qRT-PCR analysis for Stx4 mRNA expression in Cdo +/+ and Cdo −/− primary myoblasts during differentiation Full size image

Overexpression of Stx4 enhances myogenic differentiation

To investigate the function of Stx4 in myogenesis, C2C12 cells were stably transfected with control or Stx4 expression vectors and induced to differentiate. Overexpression of Stx4 in C2C12 cells generally resulted in a twofold increase of Stx4 protein (Fig. 2a) and the expression of muscle-specific genes including MHC; Myogenin and Troponin T were significantly enhanced in Stx4-overexpressing C2C12 cells, compared to that of control cells, while MyoD levels were not altered (Fig. 2b). Next, we examined the effect of Stx4 overexpression on myotube formation. Control (pcDNA) and Stx4-overexpressing C2C12 cells were induced to differentiate for 2 days, fixed, and immunostained with anti-MHC antibody followed by DAPI staining. Stx4-overexpressing C2C12 cells formed larger myotubes than the control (pcDNA) cells (Fig. 2c, d). MHC-positive cells were scored as mononucleate, containing two to five nuclei, containing six to nine nuclei, or containing ten or more nuclei. Stx4-overexpressing cells formed more larger myotubes containing six to nine nuclei (18 %) and ten or more nuclei (15 %), compared to control cells with 10 and 3 %, respectively. In contrast, the percentile of mononucleate cells decreased to 38 %, compared to 53 % of control cells (Fig. 2d). These data suggest that Stx4 promotes myoblast differentiation.

Fig. 2 Overexpression or knockdown of Stx4 promotes or blocks myoblast differentiation, respectively. a Lysates of control or Stx4-overexpressing C2C12 cells were immunoblotted with antibodies against Stx4 and pan-Cadherin as a loading control. The relative signal intensity of Stx4 to pan-Cadherin was quantified and added under the blot. b Lysates of control or Stx4 expression vector transfected C2C12 cells from the differentiation day 1 (D1) and D2 were immunoblotted with antibodies to MHC, MyoD, Myogenin, Troponin T, and pan-Cadherin as a loading control. c Control or Stx4 expression vector transfected C2C12 cells were induced to differentiate for 3 days and immunostained with MHC antibody followed by DAPI staining to visualize nuclei. Size bar = 100 μm. d The quantification of myotube formation shown in panel c. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results. Significant difference from control, *p < 0.01. e Control or shStx4 expression vector transfected C2C12 cells were analyzed by immunoblotting with antibodies to Stx4 and pan-Cadherin as a loading control. The relative knockdown levels of Stx4 to pan-Cadherin are quantified and added under the blot. f Control or shStx4 expression vector transfected C2C12 cells were induced to differentiate for 2 or 3 days and then lysates were subjected to immunoblotting with antibodies to MHC, MyoD, Myogenin, Troponin T, and pan-Cadherin as a loading control. g Control pSuper or shStx4 expression vector transfected C2C12 cells were induced to differentiate for 3 days and immunostained with an antibody to MHC followed by DAPI staining to visualize nuclei. Size bar = 100 μm. h The quantification of myotube formation shown in panel g. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results. Significant difference from control, *p < 0.01, **p < 0.005 Full size image

The depletion of Stx4 decreases myogenic differentiation

To examine whether Stx4 depletion inhibits muscle-specific gene expression and myotube formation, C2C12 cells were stably transfected with control pSuper or Stx4 shRNA (shStx4) expression vectors, induced to differentiate for 3 days and analyzed for their differentiation ability by Western blot analysis and immunostaining with anti-MHC antibody. We have tested five different Stx4 shRNA expression vectors, and among them, two shRNA constructs reproducibly resulted in a significant knockdown of Stx4. Among these, we used mostly shStx4#1 for this study (Additional file 1: Figure S1). Stx4 protein levels decreased to 34 % in C2C12/shStx4 cells, relative to that of C2C12/pSuper cells (Fig. 2e). Stx4-depleted cells exhibited a dramatic reduction in the expression of MHC, MyoD, Myogenin, and Troponin T, compared to C2C12/pSuper cells (Fig. 2f). Furthermore, C2C12/shStx4 cells formed smaller myotubes with fewer nuclei, relative to C2C12/pSuper cells (Fig. 2g). The quantification of MHC-positive cells showed that Stx depletion resulted in the formation of more mononucleated myocytes (~57 to ~67 %) and less myotubes with more than six nuclei (~17 to ~9 %), relative to C2C12/pSuper cells (Fig. 2h). These results indicate that Stx4 is required for efficient myoblast differentiation.

Stx4 and Cdo interact physically in differentiating myoblasts, and this interaction is mediated by the t-SNARE domain of Stx4

Next, we examined whether Stx4 and Cdo physically interacts in mammalian cells. To do so, 293T cells were transiently transfected with myc-tagged Stx4 and Cdo and then lysates were immunoprecipitated with the myc-tag antibody followed by immunoblotting with Cdo and myc antibodies. Consistent with the result obtained from a previous yeast two-hybrid screening [13], Stx4 and Cdo interacted in 293T cells when coexpressed (Fig. 3a). To assess whether Stx4 and Cdo interact endogenously in myoblasts, cell lysates of differentiating C2C12 myoblasts from a total of 3 days of differentiation time course were immunoprecipitated with control IgG or an anti-Cdo antibody and analyzed by Western blotting. Stx4 was precipitated with Cdo throughout differentiation time course, and the coprecipitation was highest at D2 (Fig. 3b) when myoblasts were differentiating (Fig. 1b). These results suggest that Stx4 and Cdo can physically interact in myoblasts during differentiation.

Fig. 3 Stx4 and Cdo interact physically in differentiating myoblasts, and the t-SNARE domain of Stx4 mediates the interaction with Cdo. a Lysates of 293T cells transfected with Stx4-myc, Cdo, or control vector were subjected to immunoprecipitation with myc and immunoblotting with Cdo or myc antibodies. b Lysates of C2C12 cells from various differentiation time courses were immunoprecipitated with control IgG or anti-Cdo antibody and immunoblotted with antibodies to Stx4, Cdo, and pan-Cadherin as a loading control. c The schematic representation of the domain structure of Cdo. Cdo consists of five immunoglobulin, three fibronectin type III, a single transmembrane domain, and a 270-amino-acid-long intracellular region. d 293T cells were transiently cotransfected with control or Stx4-myc along with either the full length or three deletion mutants of the Cdo’s cytoplasmic region. Forty-eight hours later, cell lysates were subjected to immunoprecipitation with myc antibody followed by immunoblotting with Cdo antibody. Total lysates served as the expression controls. e The schematic representation depicts the domain structure of Stx4 and the deletion of the specific domain; Δ33-153 (the Syntaxin domain deletion), Δ195-262 (the t-SNARE region deletion), and Δ154-194 (the linker region deletion). f 293T cells were transiently cotransfected with control or Cdo along with the full length or deletion mutants of Stx4, and the lysates were pulled down with S-agarose beads followed by immunoblotting with Cdo or GFP antibodies Full size image

Cdo consists of an extracellular region that contains five immunoglobulin (Ig)-like repeats followed by three fibronectin type III (FNIII)-like repeats, a transmembrane segment, and a long cytoplasmic tail [31]. A schematic representation of the Cdo protein structure is shown in Fig. 3c. To determine the cytoplasmic region of Cdo which is responsible for interaction with Stx4, we have transiently transfected three Cdo mutants that harbor indicated deletions in the cytoplasmic tail and analyzed the ability of these mutants to coprecipitate with Stx4. All three Cdo mutants showed a reduction in Stx4 binding, relative to the full length. However, CdoΔ986-1048 and CdoΔ1035-1160 failed to coprecipitate Stx4 (Fig. 3d), suggesting the cytoplasmic region of Cdo is required for Stx4 interaction.

To identify the domain of Stx4 responsible for Cdo interaction, we have generated GFP-S-tagged Stx4 deletion mutants based on its domain structure (Fig. 3e). The full length or these deletion mutants of Stx4 were cotransfected into 293T cells with Cdo expression vector followed by a pulldown analysis with S-agarose beads and Western blot analysis. While Stx4Δ33-153 and Stx4Δ154-194 proteins pulled down Cdo similarly to the full-length Stx4, Stx4Δ195-262 failed to precipitate Cdo, suggesting that the t-SNARE domain (aa 195–262) of Stx4 is responsible for Cdo binding (Fig. 3f).

The deletion mutants for either the Syntaxin or the t-SNARE domain of Stx4 failed to enhance myoblast differentiation

The promyogenic function of Cdo involves the activation of MyoD via p38MAPK pathway [1]. Therefore, we assessed the effect of Stx4 or/and Cdo expression on MyoD activation by using a MyoD-responsive reporter. To do so, 10T1/2 fibroblasts were cotransfected with a MyoD-luciferase construct and a MyoD expression vector along with expression vectors for Stx4 and/or Cdo. Forty-eight hours later, lysates were subjected to a luciferase assay. The expression of Stx4 or Cdo singly with MyoD enhanced the luciferase activity approximately 2.5-fold and 2.7-fold, respectively, while coexpression of Stx4 and Cdo enhanced the MyoD-reporter activity to approximately 5.8-fold compared to control MyoD-expressing cells (Fig. 4a). These data suggest that Stx4 and Cdo can activate MyoD cooperatively.

Fig. 4 Stx4 and Cdo induce MyoD activities synergistically, and the Cdo-binding deficient Stx4 mutant failed to enhance myotube formation. a 10T1/2 cells were cotransfected with a MyoD-luciferase reporter and the expression vectors for MyoD and β-galactosidase as an internal control. In addition, control, Stx4, and/or Cdo expression vectors were cotransfected as indicated. Forty-eight hours later, the reporter activities were measured and normalized relative to the internal control. The experiment was performed as triplicates and repeated three times with similar results. *p < 0.01. b Lysates of C2C12 cells stably transfected with indicated Stx4 vectors were immunoblotted with antibodies to MHC and pan-Cadherin as a loading control. The relative signal intensities of MHC to pan-Cadherin were quantified and added under the blot. c C2C12 cells were transiently cotransfected with control (pcDNA), Stx4, or Stx4 mutants along with a GFP expression vector to mark the transfectant. Then, cells were induced to differentiate for 2 days, followed by immunostaining with an antibody to MHC and DAPI stain. Size bar = 100 μm. d Quantification of myotube formation of cell lines shown in panel c. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results. Significant difference from control, *p < 0.01, **p < 0.005 Full size image

To assess the functional significance of the Stx4 interaction with Cdo in myoblast differentiation, C2C12 cells stably transfected with the control, the full length, or deletion mutants of Stx4 as indicated and induced to differentiate for 2 days followed by Western blotting for MHC expression. C2C12 cells expressing either the full-length Stx4 or Stx4Δ154-194 displayed enhanced MHC expression, compared to control-vector-expressing cells. In contrast, the expression of Stx4Δ33-153 or Stx4Δ195-262 resulted in starkly decreased MHC expression (Fig. 4b). To assess the effect of Stx4 deletion mutants on myotube formation, C2C12 cells were cotransfected with the control pcDNA, the full length, or deletion mutants of Stx4 and GFP to mark transfectants and induced to differentiate for 3 days followed by immunostaining for MHC expression. Consistent with the Western blot data, the expression of Stx4Δ154-194 enhanced myotube formation to a comparable level of the full-length Stx4, as seen by fewer GFP-positive MHC-negative cells and larger GFP-positive myotubes with more nuclei per myotube, relative to control cells (Fig. 4c, d). However, roughly 60 % of the control pcDNA, Stx4Δ33-153-, or Stx4Δ195-262-expressing cells were negative for MHC expression, and a large proportion of the GFP- and MHC-positive cells were mononucleated in these cultures. These results suggest that the Syntaxin and t-SNARE domains of Stx4 are required for the promyogenic function of Stx4.

Stx4 enhances p38MAPK phosphorylation, and Stx4 overexpression restores myoblast differentiation in Cdo-depleted cells

Previously, we have reported that Cdo promotes myoblast differentiation via activation of a key promyogenic kinase p38MAPK (p38) [13], and this is required for the efficient myoblast differentiation [13, 32]. Therefore, we examined the effect of Stx4 on p38 activation in C2C12 cells. Control or Stx4-overexpressing C2C12 cells were induced to differentiate for 2 days, and the status of p38 activation was analyzed by Western blot analysis with antibodies to an active phosphorylated form of p38 (p-p38) or total p38. Overexpression of Stx4 led to a substantial increase in p-p38 levels relative to that of control cells, while the level of total p38 was unchanged (Fig. 5a). In addition, C2C12/pSuper or C2C12/shStx4 cells were induced to differentiate for 3 days and analyzed for p38 activation. Stx4 knockdown in C2C12 cells caused a notable decrease in p-p38 levels relative to that of the control cells (Fig. 5b), suggesting that Stx4 is required for p38 activation during myoblast differentiation. We next asked whether the decreased p38 activation in Cdo-depleted C2C12 cells can be rescued by Stx4 expression. C2C12/pSuper and C2C12/shCdo cells were transiently transfected with pcDNA or Stx4 expression vector, plus GFP expression vector to label the transfectants. After 2 days of transfection, cells were immunostained with antibodies to p-p38 and GFP followed by DAPI staining to visualize nuclei. The representative pictures are shown in Fig. 5c. Roughly 36 % of control-transfected C2C12/pSuper cells were positive for the nuclear p-p38 accumulation (marked with a white arrow), whereas 63 % of Stx4-transfected C2C12/pSuper cells were positive for p-p38. On the other hand, only 17 % of control-transfected C2C12/shCdo cells were weakly positive for p-p38, whereas Stx4-expressing C2C12/shCdo cells displayed restored p-p38 levels with 31 % which is similar to control-transfected C2C12/pSuper cells (Fig. 5d). The quantification of relative p-p38 signal intensities in GFP-positive cells revealed the increased signal intensity in Stx4-overexpressing control cells. Furthermore, the overexpression of Stx4 restored p38 activation in C2C12/shCdo cells (Fig. 5e).

Fig. 5 Overexpression of Stx4 can override the block of myoblast differentiation in Cdo-depleted cells. a, b C2C12 cells were transfected with control, Stx4, or shStx4 expression vectors, and the lysates were analyzed for the levels of phospho-p38MAPK (p-p38) relative to total p38MAPK (p38). The relative levels of p-p38 are quantified and added under each well. c C2C12/pSuper and C2C12/Cdo shRNA cells were transiently transfected with pcDNA or Stx4 expression vector, plus GFP expression vector to mark transfectants. Confluent cultures were then fixed and stained with antibody to p-p38 (red). Cell nuclei were visualized by staining with DAPI (blue). The white arrows in p-p38 panels mark the transfected cells. Size bar = 10 μm. d Quantification of cultures shown in c. GFP+ cells were scored as positive or negative for p-p38 staining. These experiments were repeated three times with similar results. Significant difference from control, *p < 0.01. e Quantification of the relative signal strength of p-p38 in GFP-positive cells. Values are determinants of 10 fields and experiments were repeated three times with similar results. *p < 0.01. f C2C12/pSuper and C2C12/Cdo shRNA cells were transiently transfected with pcDNA or Stx4 expression vector, and the lysates were analyzed for the levels of phospho-p38MAPK (p-p38) relative to total p38MAPK (p38). The relative levels of p-p38 are quantified and added under each well. g C2C12 cells were transfected with pSuper or shStx4 expression vectors and were immunoprecipitated with control IgG or anti-Cdo antibody and immunoblotted with antibodies to JLP, Bnip2, Cdo, Stx4, and pan-Cadherin as a loading control. h Similar sets of cells as shown in panel c were induced to differentiate for 3 days and immunostained for MHC and GFP expressions. Size bar = 100 μm. i Quantification of myotube formation from panel e. GFP+ myotubes were quantified for MHC expression and the number of nuclei present in myotubes. Values are determinants of more than 10 fields and these experiments were repeated three times with similar results. Significant difference from control, *p < 0.01, **p < 0.005 Full size image

To further confirm these results, C2C12/pSuper and C2C12/shCdo cells were transfected with control or Stx4 expression vectors and induced to differentiate for 2 days, followed by Western blot analysis. As expected, C2C12/pSuper cells overexpressing Stx4 displayed elevated p-p38 levels while control transfected Cdo-depleted cells showed a reduction in p-p38 levels. In consistent with the aforementioned data, Stx4 expression in C2C12/shCdo cells restored p38 activation (Fig. 5f). Since Cdo can activate AKT via interaction with APPL1 in promotion of myoblast differentiation [15], we next examined the effect of Stx4 on AKT activation in C2C12 cells. However, the active phosphorylated AKT levels were not altered by depletion or overexpression of Stx4 (Additional file 2: Figure S2). These data suggest that Stx4 overexpression can override the block of p38MAPK activation caused by Cdo depletion in C2C12 myoblasts.

To further examine the role of Stx4 in Cdo-mediated p38 activation, we have assessed the effect of Stx4 depletion on the complex formation of Cdo with Bnip2 and JLP which has been shown to be critical for p38 activation and myogenic differentiation [13, 14]. C2C12 cells were transfected with control or shStx4 expression vector and induced to differentiate for 2 days. Cell lysates were subjected to immunoprecipitation with control IgG or anti-Cdo antibody followed by immunoblotting. The interaction of Cdo with JLP, Bnip2, and Stx4 was abrogated in Stx4-depleted cells compared to control cells (Fig. 5g). Interestingly, Cdo levels in total lysates were slightly decreased in Stx4-depleted C2C12 cells, while the levels of JLP and Bnip2 were not altered. These data suggest that Stx4 is required for Cdo/Bnip2/JLP complex formation.

This led us to investigate whether overexpression of Stx4 can restore the differentiation ability of Cdo-depleted myoblasts. C2C12/pSuper and C2C12/shCdo cells were transiently transfected with pcDNA or Stx4 plus GFP expression vectors to label the transfectants and induced to differentiate for 3 days, followed by immunostaining with a MHC antibody and DAPI staining. Consistently, Stx4 overexpression in C2C12/pSuper cells enhanced myotube formation as seen by the increased proportion of larger myotubes containing more than six nuclei compared with the control transfected cells (Fig. 5h, i). Similarly to the previous reports [26], C2C12/shCdo cells transfected with the control pcDNA exhibited impaired myotube formation. Overexpression of Stx4 in these cells restored myotube formation to similar levels of control cells (Fig. 5h, i). These results demonstrate that overexpression of Stx4 can restore the differentiation ability of Cdo-depleted C2C12 myoblasts.

Depletion of Stx4 causes a reduction in Cdo protein levels at the cell surface

Next, we examined whether Stx4 regulated Cdo translocation to cell surface. To do so, we have assessed whether Stx4 depletion altered the level of Cdo at the cell surface by surface biotinylation. Stx4 knockdowned C2C12 cells displayed decreased Cdo protein levels at the cell surface as well as total Cdo proteins in lysates (Fig. 6a). Furthermore, this effect on Cdo levels appears to be specific since N-Cadherin levels did not alter in these cells. To further examine, C2C12/pSuper or C2C12/shStx4 cells were transfected with a Cdo-GFP vector and subjected to immunostaining with a Cadherin antibody to label the membrane and confocal microscopy. Cdo-GFP proteins were found at the cell membrane and intracellular compartments in both cell types. However, the signals of Cdo-GFP and Cadherin were partially superimposed at the membrane in control cells, whereas Cdo-GFP and Cadherin signals did not largely overlap at the membrane in Stx4-depleted cells (Fig. 6a). Next, we assessed whether the amount of Cdo at the cell surface is rescued by Stx4 in Cdo-depleted C2C12 cells. C2C12/shCdo cells were transfected with the Stx4 expression vector, and 24 h later, cells were analyzed by surface biotinylation. At this condition, Cdo was decreased in both pcDNA and Stx4-transfected C2C12/shCdo cells, compared to the control C2C12/pSuper cells (Fig. 6c). Consistently, the biotinylated Cdo levels were decreased in control C2C12/shCdo cells while Stx4 overexpression restored the biotinylated Cdo levels in C2C12/shCdo cells to the control C2C12/pSuper cells. These data suggest that Stx4 enhances Cdo translocation to the cell surface thereby stimulating Cdo-mediated p38 activation and myoblast differentiation.

Fig. 6 The cell-surface-resident Cdo was specifically decreased in Stx4-depleted C2C12 cells. a C2C12 cells were transfected with pSuper or shStx4 expression vectors, and cells at the indicated differentiation time points were subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were also analyzed as control. b C2C12/pSuper or C2C12/shStx4 cells were transfected with Cdo-GFP expression vectors and subjected to immunostaining with N-Cadherin antibody, followed by confocal microscopy. The boxed area is shown as an enlarged view. The white arrows mark the area where the localization of Cdo-GFP under the N-Cadherin-resident cell surface is located. Size bar = 10 μm. c C2C12/pSuper or C2C12/shCdo cells were transfected with pcDNA or Stx4 expression vectors and subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were analyzed as control. d Control or shCdo-transfected C2C12 cells at D1 were subjected to surface biotinylation followed by streptavidin-bead pulldown and immunoblotting with indicated antibodies. e Control or shCdo expression vector transfected C2C12 cells were immunoprecipitated with antibody to GLUT4 and immunoblotted with antibodies to GLUT4, Stx4, and Cdo. f Stable C2C12 cells transfected with control, Cdo, or shCdo expression vector were incubated with or without 10 μg/ml insulin for 1 h, followed by 2-NBDG incubation for a further 1 h. Glucose uptake was measured by the relative fluorescence intensity. The experiment was repeated for three independent assays with similar results. Significant difference from insulin-incubated cells, *p < 0.05, **p < 0.01 Full size image

Next, we have assessed whether Cdo is involved in GLUT4 trafficking to the cell surface mediated by Stx4. To do so, C2C12/pSuper or C2C12/shCdo cells were induced to differentiate for 1 day and analyzed for the surface biotinylation of GLUT4. The biotinylated GLUT4 levels were decreased in Cdo-depleted cells, while total GLUT4 levels did not alter (Fig. 6d). Interestingly, GLUT4 interaction with Stx4 was decreased in Cdo-depleted C2C12 cells, without affecting the total expression levels of these proteins (Fig. 6e). To assess the effect of Cdo on glucose uptake in C2C12 myoblasts, cells were stably transfected with control, Cdo, or shCdo expression vectors and treated with insulin and a fluorescent glucose analog 2-NBDG. Overexpression of Cdo in C2C12 cells generally resulted in a twofold increase of 2-NBDG uptake, while Cdo depletion reduced the level of 2-NBDG uptake to about 71 % in C2C12/shCdo cells, relative to that of control cells (Fig. 6f). Taken together, these data suggest that Stx4 regulates Cdo protein levels at the cell surface thereby enhancing the promyogenic signal triggered by Cdo, such as p38MAPK. In turn, this signaling appears to be critical for GLUT4 translocation to the cell membrane mediated by Stx4.