FIG. 1.

Pak4 induces senescence in primary MEFs. A. Expression and activity of ectopic Pak4 in infected cells at day 2 postselection. Primary MEFs were infected with retroviruses expressing activated PAK4 (PAK4*), empty vector (E.V.), or H-RasV12. At day 2 postselection, Myc-PAK4 protein levels were assessed by Western blotting with an antibody directed against the Myc epitope. PAK activity was verified by immunocomplex kinase assay using autophosphorylation as a marker for kinase activity. B. Pak4 levels in infected MEFs are lower than the levels in cancer cell lines. Cell extracts were prepared from MCF7 breast cancer cells and from primary fibroblasts (MEFs) infected with activated Myc-tagged Pak4. Equal amounts of protein from each sample were subject to Western blot analysis using a monoclonal antibody directed against PAK4 (upper panel) or actin (bottom panel). C. Expression of Pak4 inhibits growth of primary MEFs. Representative growth curves of primary MEFs infected with empty pLPC vector (triangles), activated PAK4* (circles), or RasV12 (squares) are shown. D. Expression of Pak1 does not inhibit growth of primary MEFs. Representative growth curves of primary MEFs infected with empty pLPC vector (triangles), activated Pak4 (PAK4*; closed circles), or activated Pak1 (PAK1*; closed squares) are shown. E and F. Decrease in BrdU incorporation in cells expressing Pak4. Percentage of BrdU-incorporating cells at day 6 postselection in cell populations infected with empty vector, activated PAK4 (PAK4*), or RasV12 is indicated. Incorporated BrdU was detected by immunofluorescence using an anti-BrdU antibody. About 200 cells from each population were scored, and the average and standard deviation for at least three experiments are shown. Typical fields of cells are shown in panel F. Left: BrdU-incorporating nuclei are stained orange with rhodamine-conjugated secondary antibody and indicated by an arrow. The cytoskeleton is stained green with fluorescein isothiocyanate-labeled phalloidin. Right: different fields of the same preparation of cells treated with BrdU and stained for both BrdU (orange) and 4′,6′diamidino-2-phenylindole (blue). Bar, 20 μm. G and H. Expression of Pak4 leads to an increase in SA-β-Gal expression. Percentages of cells positive for SA-β-Gal (at pH 6.0) at day 4 (open bars) and at day 6 (closed bars) postselection are indicated. Cells were scored as in panel E. Typical fields of cells are shown in panel H. SA-β-Gal-positive cells stain blue. I. SA-β-Gal levels are not elevated in response to Pak1 in MEFs. Percentages of cells positive for SA-β-Gal (at pH 6.0) at day 6 postselection are shown. Cells were scored as in panel E.