Figure 7.
GSK3α activation promotes endocytosis in muscle cells. (A) Effect of GSK3α mutants on transferrin internalization in myotubes. C2C12 myoblasts transfected with indicated GSK3α mutants and differentiated into myotubes were incubated with Tfn-488 at 37°C for 20 min. Cells were then washed on ice, fixed, and imaged under confocal microscopy. Scale bar, 10 µm. (B) Fluorescence intensity of internalized transferrin was quantified and shown (n = 25 cells from three independent repeats). (C) Effect of GSK3α mutants on GLUT4-HA-GFP distribution. L6 myoblasts were infected with indicated GSK3α mutants and transfected with GLUT4-HA-GFP. Scale bar, 10 µm. (D) After immunofluorescent staining with anti-HA antibody without permeabilization and imaging by confocal microscopy, the relative amount of surface GLUT4-HA-GFP was quantified by the fluorescent intensity of HA signaling in red divided by the total GLUT4-HA-GFP in green as shown (n ≥ 30 cells from three independent repeats). (E–H) Effect of GSK3 inhibitors on GLUT4-HA-GFP distribution. L6 myoblasts transfected with GLUT4-HA-GFP were treated with DMSO, GSK3α inhibitor (BRD0705, 20 µM), or GSK3β inhibitor (TWS119, 2 µM) under growth (E) or serum-free medium (G) for 3 h. Scale bar, 10 µm. The relative amount of surface GLUT4-HA-GFP was quantified by the fluorescent intensity of HA signaling in red divided by the total GLUT4-HA-GFP in green as shown in F and H (n ≥ 30 cells from three independent repeats). Data are shown as median ± min and max value. Data are analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.

GSK3α activation promotes endocytosis in muscle cells. (A) Effect of GSK3α mutants on transferrin internalization in myotubes. C2C12 myoblasts transfected with indicated GSK3α mutants and differentiated into myotubes were incubated with Tfn-488 at 37°C for 20 min. Cells were then washed on ice, fixed, and imaged under confocal microscopy. Scale bar, 10 µm. (B) Fluorescence intensity of internalized transferrin was quantified and shown (n = 25 cells from three independent repeats). (C) Effect of GSK3α mutants on GLUT4-HA-GFP distribution. L6 myoblasts were infected with indicated GSK3α mutants and transfected with GLUT4-HA-GFP. Scale bar, 10 µm. (D) After immunofluorescent staining with anti-HA antibody without permeabilization and imaging by confocal microscopy, the relative amount of surface GLUT4-HA-GFP was quantified by the fluorescent intensity of HA signaling in red divided by the total GLUT4-HA-GFP in green as shown (n ≥ 30 cells from three independent repeats). (E–H) Effect of GSK3 inhibitors on GLUT4-HA-GFP distribution. L6 myoblasts transfected with GLUT4-HA-GFP were treated with DMSO, GSK3α inhibitor (BRD0705, 20 µM), or GSK3β inhibitor (TWS119, 2 µM) under growth (E) or serum-free medium (G) for 3 h. Scale bar, 10 µm. The relative amount of surface GLUT4-HA-GFP was quantified by the fluorescent intensity of HA signaling in red divided by the total GLUT4-HA-GFP in green as shown in F and H (n ≥ 30 cells from three independent repeats). Data are shown as median ± min and max value. Data are analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.

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