p53 Response to Senescence Figure 5. A) Oncogenic Ras increased PPP1CA protein levels. P53ts (control) or p53ts-Ras (+ras) cells were grown at 39uC or incubated at 32uC for 24 hrs. Then PPP1CA protein levels were analyzed by western Blot. a-tubulin was used as a loading control. The data are representative of three independent experiments. B) PPP1CA mRNA levels were not dependent on the expression of oncogenic ras. mRNA levels were analyzed by Northern blot. A labeled probe able to specifically recognize PPP1CA isoform was used as described in M&M. C) Oncogenic ras increased PP1 activity. Exponentially growing cells were keep growing or switch for 24 hrs at 32uC as indicated. Then were starved and PP1 phosphatase activity was measured as described in M&M. C shows the remaining activity after 100 nM okadaic acid treatment to inhibit PP1 and PP2A activity. D, E, and F) P53ts-Ras cells carrying the shRNA against PPP1CA (shRNA) or vector alone (control) were grown at restricted temperature (39uC), or permissive temperature (32uC) as indicated, for 24 hrs. Cells were harvested for protein extraction (for D), fixed and stained with crystal violet (for E) or for SA b- GAL (for F). D) Downregulation of PPP1CA inhibits p53-induced pRb hypophosphorylation. Cells were processed for western blot, showing hyperphosphorylated (ppRb) and hypophosphorylated (pRb) forms of the protein. a-tubulin was used as a loading control. The data are representative of three independent experiments. Bottom panel shows quantification of pRb bands. E) and F) Downregulation of PPP1CA bypasses p53/ras-induced senescence. Cells (104) were seeded and grown at 39u or 32uC for 1 week, then fixed and stained for colony formation with crystal violet (E) or SA b-GAL (F). In F, numbers show the percentage of cells with SA b-GAL staining. doi:10.1371/journal.pone.0003230.g005 PLoS ONE | www.plosone.org 9 September 2008 | Volume 3 | Issue 9 | e3230