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. 2018 Jun 12;32(12):6445–6455. doi: 10.1096/fj.201701151RRR

Figure 3.

Figure 3

GlcNAc promotes myogenesis. A) Regulation of the synthesis of Asn-linked glycans by GlcNAc in muscle cells. Glucosamine is taken up by a glucose transporter. Most glucosamine is used for glycolysis and glycogen synthesis, and only a small percentage is transformed into UDP-GlcNAc. In contrast, extracellular GlcNAc is rapidly endocytosed to synthesize UDP-GlcNAc. UDP-GlcNAc is then incorporated into Asn-linked glycans, which are attached to proteins in the Golgi apparatus. The glycoproteins are then transported to the cell surface. Galectin-3 binds to the glycoproteins. B) Myogenesis was induced for 62 h in the presence or absence of GlcNAc. The number of nuclei in MHC+ multinucleated myotube was counted, and the fold increase in myogenesis was calculated taking cells treated with PBS(−) as 1. Each data point represents the mean of the fold increase obtained in 3 independent experiments, with error bars indicating sd. Significance compared with control (0 mM) by 1-way ANOVA is shown. **P < 0.01. When ANOVA–on ranks was used, the detected significance was as follows: GlcNAc, 0.2 mM and 1 mM, where P < 0.05. C) Myogenesis was induced in the presence or absence of saccharides (Gal and mannose) at a concentration of 10 mM. The total areas of MHC+ multinucleated myotubes were calculated, and the fold increase in the myogenesis was calculated taking cells treated with PBS(−) as 1. Each data point represents the mean of the fold increase obtained in 3 independent experiments with error bars indicating sd. Significance compared with control by 1-way ANOVA is shown. **P < 0.01. When ANOVA–on ranks was used, the detected significance was the following: Gal, where P < 0.05. D, E) Stitched, extended-focus images (5 × 5 FOVs) of myotubes (MHC+ cells are white) formed in the absence (D) or presence (E) of GlcNAc after differentiation for 72 h. Representative images from 3 independent experiments are shown. F) Myogenesis was induced for 72 h, and the number of nuclei in each MHC+ multinucleated myotube found in the stitched FOVs was counted and categorized into 3 groups based on the number of nuclei in each myotube. The representative results obtained from 2 independent experiments are shown. G) Myogenesis was induced in the presence of GlcNAc (1 mM) together with different concentrations of the galectin-3 antagonist, lactose. The total areas of MHC+ multinucleated myotubes were calculated and the fold increase in the myogenesis was calculated taking cells treated with GlcNAc as 1. Each data point represents the mean of the fold increase obtained by 3 independent experiments with error bars indicating sd. Significance compared to control by 1 way -ANOVA is shown. **P < 0.01. When ANOVA–on ranks was used, the detected significance was the following: lactose 10 mM, where P < 0.05.