Figure 3. Pharmacological inhibition of PTEN drives senescence in vitro and in vivo.

(A) Quantification of the senescence-associated β-gal assay and its staining in Pten^+/+ (WT) and Pten^+/– (HET) cells treated with indicated concentrations of the PTEN inhibitor VO-OHpic. Scale bar: 10 μm. Error bars show SD. (B) Western analysis of cells from A treated with 500 nM VO-OHpic. Blot lanes were run on the same gel but were noncontiguous. (C) Quantification of pAkt (Ser 473)/Akt protein levels in Pten^+/+ and Pten^+/– MEFs from A, normalized for the WT baseline level (dashed line). Error bars show SD from independent experiments. (D) Quantification of senescence-associated β-gal staining in Pten-null MEFs treated with either vehicle or 500 nM VO-OHpic. Error bars show SD. (E) Western analysis for PTEN and its quantification in 6 prostate cancer cell lines with WT (black bars) and mutant (red bars) p53. Error bars show SD from independent experiments. (F) Fold increase in tumor volume in a MDA PCa 2b xenograft mouse model after systemic treatment with VO-OHpic. Error bars show SD. A representative Western blot analysis for p53 in tumors from mice treated with Vehicle or VO-OHpic is shown in the inset. Numbers in the inset indicate densitometrically determined protein levels for p53 relative to β-actin (G) Quantification of β-gal– and Ki-67–positive cells in MDA PCa 2b tumors, untreated and treated with VO-OHpic. Error bars show SD. P indicates the statistical significance as measured by Student’s t test throughout.