Fig. 4.

a Effect of high glucose and high insulin combination (HGHI glucose concentration 15 mmol/l insulin concentration 50 nmol/l) and insulin-like growth factor-I (IGF-I, concentration 30 nmol/l) on cyclin D1 (Cyc D1) in C2C12 myogenic cell cultures. The level of Cyc D1 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 in appropriate 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. appropriate control (Ctrl) for the same parameter and the same culture conditions. Hash symbol indicates significantly different vs. high glucose and high insulin effect for the same parameter. b Cellular content and localization of cyclin D1 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 Cyc D1 (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. The IOD values of Cyc D1-related green fluorescence were evaluated using MicroImage analysis system (Olympus Optical Poland). The data were presented as mean ± SD with n = 10/treatment conditions of total fluorescence of fields with similar cell density and as a nucleus/cytoplasm immunofluorescence ratio. Asterisk indicates significantly different vs. control (Ctrl) value. c The level of cyclin D1 bound to cyclin-dependent kinase 4 (Cdk4) in proliferating and differentiating myogenic cells. The presence of Cyc D1-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 were precipitated and recovered from whole cell lysate. Blots are representative of three separate experiments