FIG. 3.

Activation of MEKK3 inhibits DNA synthesis. NIH 3T3 [MEKK3-ER] cells were starved in the absence of serum for 3 days with or without E2 and then stimulated with 10% FCS in the presence of 10 μM BrdU (with or without E2). (A) Cells were fixed and analyzed by indirect immunofluorescence for BrdU incorporation, shown in red. Nuclei were visualized with DAPI (blue). (B) Cells were processed for FACS analysis to quantitatively measure BrdU incorporation (% BrdU) and DNA content, the latter to determine the percentage of cells in different phases of the cell cycle. Similar results were obtained in two independent experiments. As demonstrated by both indirect immunofluorescence and FACS analysis, the number of cells actively synthesizing DNA is greatly reduced in the presence of active MEKK3.