Figure 4

GSK-3 inhibition by LiCl blocks glucocorticoid-induced inhibition of myogenesis. Differentiating C₂C₁₂ myoblasts were either cultured in the presence or absence of dexamethasone (Dex) (10 μM), LiCl (10 mM) or vc (DMSO) for 72 h after which the cells were assessed for (A) morphological changes by staining with May-Grünwald Giemsa (40× magnification, scale bar = 500 μm). (B) Myoblast fusion was quantified by determining the nuclear distribution of 1900 or more nuclei for each separate condition. The data is expressed as a percentage of nuclei residing in cells containing 1, 2, or > 2 nuclei; reflecting mononucleated myoblasts (1 nucleus), dividing or fusing myoblasts (2 nuclei) or myotubes (> 2 nuclei), respectively. (C) Next, protein levels of MyHC-f, MyLC-1, MyLC-3 and GAPDH were determined in whole cell lysates by Western blot analysis, (D) and MCK activity was measured spectrophotometrically, expressed as specific enzyme activity (units / mg protein). (E) Alternatively, differentiating C₂C₁₂ myoblasts containing a stable genomically integrated troponin I (TnI) luciferase reporter construct were cultured for 72 h in the presence or absence of LiCl (10 mM), or Dex (10 μM) or vc (DMSO). Subsequently, lysates were prepared to measure luciferase activity (RLU/mg total protein). All data shown are representative of 3 independent experiments (means ± SEM, n = 3). *p < 0.05 compared with vc control; # p < 0.05 refers to a difference between indicated conditions.