GSI Treatment Preserves Protein Synthesis in C2C12 Myotubes

Figures

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  Figure 1. Rapamycin ablates GSI-mediated elevations in myotube formation. (A) Indices of myotube fusion and area. Graph order, top left to right: Fused nuclei per field, Non-fused nuclei per field, and Total nuclei per field. Graph order, middle left to right: Myotube number per field, Nuclei per myotube per field, Fusion index per field. Graph order, bottom left to right: Myotube area (µm) per field, Area (µm) per myotube per field, Myotube area (µm) per nuclei per field. (B) Representative image of 96-h myotubes co-stained with myosin heavy chain (MHC:red) and DAPI:blue. Images were taken at 20× magnification and the scale bar = 50 µm. At the onset of differentiation C2C12 cells were treated every 12 h with either control (Con), 4 µM γ-secretase inhibitor (GSI), 100 nM rapamycin (RAP), or GSI + RAP co-treatment. All data were analyzed using a one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. Con; #### p < 0.0001 vs. GSI (n = 3 experiments). Data are mean ± SD.

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  Figure 2. GSI treatment rescues protein synthesis in the presence of rapamycin. Representative western blotting and quantification (expressed as fold change vs. control (Con) for (A) Puromycin/β-Actin (B) Phospho (p)-mTOR Ser2448/Total mTOR; (C) p-4EBP1 Thr37/46/Total 4EBP1; (D) p70S6K Thr389/Total p70S6K; and (E) p-eEF2 Thr56/Total eEF2. Next, 72 h post-differentiation, C2C12 cells were treated every 12 h with either Con, 4 µM γ-secretase inhibitor (GSI), 100 nM rapamycin (RAP), or GSI + RAP co-treatment until 96 h post-differentiation. Then, 30 min prior to collection, all cells were treated with 1 µM puromycin. All data were analyzed using a one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. Con; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. GSI; $ p < 0.05 vs. RAP (n = 3 experiments). Data are mean ± SD.

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  Figure 3. API-1 ablates GSI-mediated elevations in myotube formation. (A) Indices of myotube fusion and area. Graph order, top left to right: Fused nuclei per field, Non-fused nuclei per field, and Total nuclei per field. Graph order, middle left to right: Myotube number per field, Nuclei per myotube per field, Fusion index per field. Graph order, bottom left to right: Myotube area (µm) per field, Area (µm) per myotube per field, Myotube area (µm) per nuclei per field. (B) Representative image of 96-h myotubes co-stained with myosin heavy chain (MHC:red) and DAPI:blue. Images were taken at 20× magnification and the scale bar = 50 µm. At the onset of differentiation C2C12 cells were treated every 12 h with either control (Con), 4 µM γ-secretase inhibitor (GSI), 10 µM 4-Amino-5,8-dihydro-5-oxo-8-β-D-ribofuranosyl-pyrido[2,3-d]pyrimidine-6-carboxamide (API-1), or GSI + API-1 co-treatment. All data were analyzed using a one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. Con; #### p < 0.0001 vs. GSI (n = 3 experiments). Data are mean ± SD.

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  Figure 4. GSI treatment rescues protein synthesis in the presence of API-1 and rapamycin. Representative western blotting and quantification (expressed as fold change vs. control (Con) for (A) Puromycin/β-Actin; (B) Phospho (p)-AKT Thr308/Total AKT; (C) p-AKT Ser473/Total AKT; (D) p-mTOR Ser2448/Total mTOR; (E) p-GSK3β Ser9/Total GSK3β; (F) Active β-Catenin/β-Actin. Then, 72 h post-differentiation, C2C12 cells were treated every 12 h with either Con, 4 µM γ-secretase inhibitor (GSI), 10 µM 4-Amino-5,8-dihydro-5-oxo-8-β-D-ribofuranosyl-pyrido[2,3-d]pyrimidine-6-carboxamide (API-1), or GSI + API-1 co-treatment until 96 h post-differentiation. All cells were treated with 1 µM puromycin 30 min prior to collection. Representative western blotting and quantification for (G) Puromycin/β-Actin. Next, 72 h post-differentiation, C2C12 cells were treated every 12 h with either Con, 4 µM GSI, 100 nM rapamycin (RAP) + 10 µM API-1, or GSI + RAP + API-1 co-treatment until 96 h post-differentiation. All cells were treated with 1 µM puromycin 30 min prior to collection. All data were analyzed using a one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p…