Figure 4

Mdm2 knockdown in estrogen-treated MCF-7 cells inhibits cell proliferation. Clonal MCF-7 cell lines with mdm2 shRNA or control vector were treated with 2 μg/ml doxycycline (DOX) for three days to induce shRNA expression, followed by 10 nM estrogen (E2) for five days in the presence of DOX. (a) Western blot analysis of Mdm2, p53, p21 and Actin protein levels from whole cell lysates. (b) Number of cells was determined by Guava Viacount assay. 10,000 cells were seeded at beginning of treatments. (c) Fluorescence activated cell sorting (FACS). Cells were harvested, fixed in 30% ethanol, and cellular DNA was stained with propidium iodide. (d) Western blot analysis of Bcl-2 and Actin protein levels from whole cell lysates. (e) MCF-7 cells, grown in matrigel for three weeks, formed mass structures of three sizes: large, intermediate and small. (f) MCF-7 cells, grown in matrigel for three weeks in the presence of 10 nM estrogen and in the absence or presence of 2 μg/ml doxycycline, were fixed and stained with propidium iodide. Masses of different sizes (large, intermediate and small) were counted and presented as percent of the total population. On average, a total of 300 masses were counted. Averages of three independent experiments are shown. The significance in the percent change of large and small masses was determined by the Student t-test (P < 0.05).