Fig. 6.

Effect of high glucose and high insulin combination (HGHI, glucose concentration 15 mmol/l insulin concentration 50 nmol/l) on p21 in C2C12 myogenic cell cultures. a Cellular content and localization of p21 in mouse C2C12 myoblasts proliferating for 24 h in 2 % FBS/DMEM (Control, Ctrl) or in the presence of HGHI. Cell cultures were stained with antibodies against p21 (green), and simultaneous nuclear staining with 7-AAD (red) was performed. Images are representative of ten independent fields in three separate experiments. Bar 20 μm. b The level of p21 in whole cell lysates of proliferating myoblasts (“Proliferation”) or in 72 h after induction of myogenesis (“Differentiation”) was evaluated by immunoblotting. The densitometric quantitation of the specific bands (IOD integrated optical density) is presented in arbitrary units with the value obtained for each isoform (Bound and Free) in control (Ctrl) proliferating cells set as 100 %. As three separate experiments gave a similar pattern of results, all data within each group were combined to calculate mean ± SD with n = 9/treatment conditions, and representative blots were presented. Asterisk indicates significantly different vs. control (Ctrl) for the same p21 isoform and the same culture conditions. c The level of p21 bound to cyclin-dependent kinase 4 (Cdk4) in proliferating and differentiating myogenic cells. The presence of p21-cdk4 complexes was visualized by immunoprecipitation with the antibody specific for cdk4. The control probing with the antibody used for immunoprecipitation was also performed, to ensure that equal amounts of protein was precipitated and recovered from whole cell lysate. Blots are representative of three separate experiments