Effect of metformin on cell cycle progression and cell cycle regulatory proteins in MCF7 cells. A. Untreated cells (Con) or cells treated with 8 mM metformin for 1.5 days (Met) were stained with propidium iodide and then analyzed by flow cytometry to estimate the number of cells in each phase of the cell cycle. The experiment was repeated 3 times and the mean and standard error for each cell phase is indicated in the table. B. Equal amounts of protein from untreated cells (Con) or cells treated with metformin for 1.5 days (Met) were analyzed by western blotting using antibodies that recognize the indicated cell cycle regulatory proteins. β-Actin was detected as a loading control C. Total RNA was isolated from untreated cells (C) or cells treated with metformin (8 mM) for 1.5 days (M) and RNase protection assays were performed to detect the mRNAs encoding the indicated cyclins (left panel). Cyclin D1 mRNA levels were normalized using L32 mRNA levels (right panel). The mean of 3 independent experiments is shown and error bars indicate standard deviation. In a t test the value for the metformin treated cells was significantly different from the control with a P value of 0.006. D. MCF7 cells were treated with (Met) or without (Con) metformin (8 mM) and then extracts were prepared for immunoprecipitation (IP) using either a control antibody (C) or an anti-CDK2 antibody (CDK2). The immunoprecipitated proteins were analyzed by western blotting using antibodies that recognize CDK2, p27Kip1 or p21Cip1 as indicated on the right.