Figure 4

Overexpression of p27 ^Kip1 in the metformin-resistant MDA-MB-231 cell line leads to metformin sensitivity. A. (Left panel) MCF7 and MDA-MB-231 cells were treated with (Met) and without (Con) metformin (8 mM) for 2 days. Western blotting was used to detect phospho-AMPK, total AMPK, p27^Kip1, and β-actin. (Right panel) Same as A except that cell extracts were used to detect phospho-Rb (Serine 795), cyclin D1 or β-actin. B. Extracts from untreated MCF7 and MDA-MB-231 cells were used for western blotting of p27^Kip1, p21^Cip1, and β-actin. C. MDA-MB-231 cells were stably transfected with a construct encoding p27^Kip1 that was tagged with a protein C epitope to derive the cell line MDA-MB-231WTp27. Extracts from the parental cell line (231) and the stably transfected cell line (231WTp27) were used for western blotting to detect the epitope-tagged p27^Kip1 (anti-Protein C epitope), p27^Kip1 (p27), and β-actin. D. MDA-MB-231 cells or MDA-MB-231WTp27 cells were treated with (dashed lines) or without (solid lines) metformin (8 mM) for up to 3 days. At the indicated time points cell number was determined using a hemocytometer. Each data point represents the mean cell number from 3 independent cultures and error bars represent standard deviation. E. MDA-MB-231 cells were transiently transfected with a construct encoding p21^Cip1 or empty vector. They were then treated with metformin for the indicated time and cell number was determined as describe in D.