Fig 4. Metformin perturbs the expression of cyclins and CDKIs.

(a) Western blot analysis of p27, p21, cyclin D3 and p130 in total protein lysate from metformin-treated and control C2C12 cells. When cells reached ~ 60% confluence, were induced to differentiate by serum deprivation and treated in differentiation medium with 100μM, 2mM and 10mM metformin for 3 days. Metformin was refreshed every 24h and crude cell protein extracts were analyzed by SDS PAGE. Tubulin and vinculin were used as loading controls. Numbers above each lane represent the densitometric analysis (b) Cell cycle analysis by DNA staining with propidium iodide. C2C12 cells were cultured for 3 days in GM and treated with 100μM, 2mM and 10mM metformin (c) Flow cytometry analysis of the three different phases of the cell cycle (G0/G1, S, G2/M) is represented by the histogram graph. Quantification of the different phases of the cell cycle was performed after three independent biological replicate experiments (d) Western blot analysis of the G2/M markers cyclin B and Cdc2 kinase in total protein lysate from metformin-treated and control C2C12 cells. Cells were induced to differentiate by serum deprivation and treated with 100μM, 2mM and 10mM metformin for 3 days. Metformin was added fresh every 24h and crude cell protein extracts were analyzed by SDS PAGE. GAPDH and vinculin were used as loading controls. Numbers above each lane represent the densitometric analysis (e) Quantitation of cyclin B and Cdc2 levels after 3 days of metformin treatment. Statistical significance was evaluated by the Student’s t-test (*p<0.05).