Figure 4. mTOR is essential for senescence upon Pten loss.

(A) Western blot analysis of primary Pten^lx/lx MEFs after rapamycin treatment and acute inactivation of Pten with Ad-Cre (Pten^Δ/Δ), according to the experimental scheme shown in Supplemental Figure 1E. Numbers indicate densitometrically determined protein levels relative to β-actin. Senescence-associated β-gal staining and its quantification is also shown. Scale bar: 10 μm. Error bars show SD. (B) Western analysis in Pten-deficient and Pten-mTOR compound mutant primary MEFs (by retroviral infection/selection). Numbers indicate densitometrically determined protein levels for p53 relative to β-actin. Senescence-associated β-gal staining and its quantification is also shown. Scale bar: 10 μm. Error bars show SD. (C) Western analysis of Pten^–/–p19Arf^–/– compound mutant MEFs (by retroviral infection/selection) and quantification of p53 levels. Error bars show SD of 3 independent experiments. (D) MEFs as in C, treated with rapamycin (rapa) or DMSO for 24 hours. Error bars show SD of 3 independent experiments. (E) Western blot analysis and quantification for Pten and p53 protein levels of MEFs infected as in A and treated with MG132 48 hours after infection. Error bars show SD of 3 independent experiments. (C–E) Numbers within and above columns indicate the average p53 levels observed in independent experiments. (F) Senescence-associated β-gal staining and quantification of Pten^Δ/Δ MEFs treated with rapamycin and/or Nutlin-3 during Ad-Cre–mediated PICS. Scale bar: 10 μm. Error bars show SD of 3 independent experiments. P indicates the statistical significance as measured by Student’s t test throughout.