O-GlcNAcylation modification of MyoD regulates skeletal muscle fiber differentiation by antagonizing the UPF1 pathway

Figures

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  Figure 1. O-GlcNAcylation modification regulates the myoblast differentiation process. A–C, RT-qPCR method was employed to analyze the gene expression of Ogt, MyoD, and myosin subtypes following the addition of Flag-OGT. D–E, Western blot analysis was performed to detect the expression of OGT, O-GlcNAc, MyoD, and myosin subtypes following the addition of Flag-OGT. a and b, the analysis diagram of Western blot. F–H, RT-qPCR method was used to detect the gene expression of Ogt, MyoD, and myosin subtypes after adding sh-OGT. I–J, Western blot was used to detect the expression of OGT, MyoD, and myosin subtypes after adding sh-OGT. a and b, the analysis diagram of Western blot. K–N, immunofluorescence method was employed to detect Myh2 (red), Myh4 (green), and Myh7 (green) in C2C12 myoblasts, and the scale was 200 μm. The data are means ± s. d. (n = 3/4). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 as determined by using a t test. Myh2, myosin heavy chain 2; Myh4, myosin heavy chain 4; Myh7, myosin heavy chain 7; MyoD, myogenic differentiation factor; OGA, O-linked N-acetylaminoglucosidase; O-GlcNAc, O-linked N-acetylglucosamine; OGT, O-linked N-acetyl glucosamine transfer.

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  Figure 2. Inhibition or enhancement of O-GlcNAcylation modification can regulate the myoblast differentiation process.A–C, RT-qPCR method was used to detect the gene expression of Ogt, MyoD, and myosin subtypes after the addition of OSMI-1. D, Western blot was utilized to detect the expression of OGT, O-GlcNAc, MyoD, and myosin subtypes after the addition of OSMI-1. a–d, the analysis diagram of Western blot. E–G, RT-qPCR method was used to detect the gene expression of Ogt, MyoD, and myosin subtypes after adding Thiamet G; H, Western blot was used to detect the expression of OGT, O-GlcNAc, MyoD, and myosin subtypes after adding Thiamet G. a–d, the analysis diagram of Western blot. The data are means ± s. d. (n = 3/4). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as determined by using a t test. Myh2, myosin heavy chain 2; Myh4, myosin heavy chain 4; Myh7, myosin heavy chain 7; MyoD, myogenic differentiation factor; OGA, O-linked N-acetylaminoglucosidase; O-GlcNAc, O-linked N-acetylglucosamine; OGT, O-linked N-acetyl glucosamine transfer.

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  Figure 3. OGT antagonizes with UPF1 to suppress the ubiquitination degradation pathway of MyoD via the K48 site of MyoD.A, LC-MS/MS detection of proteins interacting with OGT in C2C12 cells. B, Co-IP method was used to detect the endogenous interaction between OGT and MyoD. C, pulldown method was used to detect the exogenous interaction between OGT and MyoD. D, pulldown method was used to detect the O-GlcNAc glycosylation modification of MyoD by OGT enzyme activity. E, detection of MyoD stability by CHX. F, detection of MyoD degradation pathway by MG132 and CQ. G, pulldown method was used to detect the regulatory effect of OGT and UPF1 on MyoD ubiquitination. H, pulldown method to detect the relationship between OGT and UPF1. I, pulldown method was used to detect the ubiquitination site of MyoD. MyoD, myogenic differentiation factor; OGT, O-linked N-acetyl glucosamine transfer; UPF1, up-frameshift protein 1; CHX, cycloheximide.

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  Figure 4. Inhibition of OGA and overexpressing MyoD can regulate the type of MyHC in the myogenic differentiation of C2C12 cells.A–E, RT-qPCR method was used to detect the gene expression of different subtypes of Oga, MyoD, and myosin. F, Western blot method was used to detect the expression of various protein subtypes, including OGA, MyoD, O-GlcNAc, and myosin. a–f, the analysis diagram of Western blot. G–J, the expression of Myh2 (red), Myh4 (green), and Myh7 (green) in C2C12 myoblasts was detected by immunofluorescence, and the scale was 200 μm. The data are means ± s. d. (n = 3/4). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as determined by using a two-way ANOVA. Myh2, myosin heavy chain 2; Myh4, myosin heavy chain 4; Myh7, myosin heavy chain 7; MyoD, myogenic differentiation factor; OGA, O-linked N-acetylaminoglucosidase; O-GlcNAc, O-linked N-acetylglucosamine; OGT, O-linked N-acetyl glucosamine transfer.

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  Figure 5. MyoD regulates the development and differentiation of skeletal muscle fibers in Ogt-deficient mice.A, breeding scheme involving mice with a specific knockout of the Ogt gene (Ogt KO) and wildtype control (WT). B–C, immunohistochemical results of mouse gastrocnemius muscle. D, RT-qPCR method was used to detect the gene expression of Ogt.E, RT-qPCR method was used to detect the gene expression of MyoD. F, Western blot was used to detect the protein expression of OGT, MyoD, and O-GlcNAc. a–c, the analysis diagram of Western blot. G–H, ATPase staining results of mouse gastrocnemius muscle tissue. I–K, RT-qPCR method was utilized to detect the gene expression of various myosin subtypes. L, Western blot analysis was employed to detect the expression of different myosin subtypes. a–c, the analysis diagram of Western blot. M–N, the expression of Myh4 (red) and Myh7 (green) in mouse gastrocnemius was identified through immunofluorescence, with a scale of 50 μm. The data are means ± s. d. (n = 3/4). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as determined by using a two-way ANOVA. Myh2, myosin heavy chain 2; Myh4, myosin heavy chain 4; Myh7, myosin heavy chain 7; MyoD, myogenic differentiation…

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  Figure 6. Mechanism of O-GlcNAcylation modification of MyoD in regulating skeletal muscle fiber differentiation.A, Wild type mice. B, Ogt gene deficient mice. MyoD, myogenic differentiation factor; OGT, O-linked N-acetyl glucosamine transfer.

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