FIG. 3.

Kip2 controls proliferation in T6PNE cells. A retroviral vector expressing green fluorescent protein (GFP) alone (A) or expressing GFP and Kip2 (B) was used to infect T6PNE cells in the absence of tamoxifen. Cell cycle status of GFP-positive cells was assessed by BrdU staining (red). No GFP-BrdU double-positive cells were found in wells infected with the bicistronic GFP-Kip2 vector (B), whereas many cells infected with the GFP control vector did take up BrdU (arrows in A). DAPI is shown in blue. To inhibit Kip2 expression, T6PNE cells were transfected with a plasmid encoding an shKip2 (D) or scrambled (C) RNA along with GFP. The efficacy of the Kip2 shRNA construct was assessed by Western blot analysis (E). In T6PNE cells in which growth was inhibited by tamoxifen (4 μM)–mediated induction of E47, transfection of the shKip2 but not control shRNA (visualized by co-coexpression of GFP) caused cell cycle reentry as determined by colocalization of BrdU (red) and GFP (these are green cells with yellow nuclei; 6 such cells are marked by white arrows in D).