(A) PCR analysis to detect recombination of mTor^Δ allele on DNA from several mouse tissues. AP, VP and DLP stand for anterior prostate, ventral prostate and dorsolateral prostate respectively. (B) mTor quantitative RT-PCR analysis of the prostate mRNA from WT and mTor^pc−/− mice. Error bars show S.D. from three independent experiments. *, P < 0.05. (C) mTor genomic quantitative real-time PCR on DNA extracted from laser capture micro-dissected (LCM) epithelial cells of WT, mTor^pc+/− and mTor^pc−/− mice prostates. The inset shows the genotyping by PCR of the DNA extracted from the prostatic epithelial cells obtained by LCM and subsequently analyzed by genomic quantitative real-time PCR. (D) Representative images of prostates from mTor^pc−/− and wild-type (WT) 3 month old mice. (E) Weights of prostate from mTor^pc−/− and WT mice. Error bars show S.D. in the weight of 5 mice from each genotype. (F) Hematoxylin/Eosin (H&E) staining of AP, VP and DLP sections from the same mice showed in (D). (G) Upper panel: cell suspensions from mTor^pc−/− and WT mouse prostates stained with lineage markers (CD31, CD45, Ter119) and for α6-integrin (CD49f). Gates indicate the Lineage negative, α6-integrin positive, basal epithelial cell population. Lower panel: representative overlay histogram of mTor^pc−/− and WT basal epithelial cell Forward Scatter (FSC), an indicator of cell size. *, P < 0.05. (H) Size of the litters from WT and mTor^pc−/− males at 3 months of age. There is not significant difference between the litters from WT and mTor^pc−/− males.

(A) MRI analysis of prostatic tumors (dashed yellow circles) in Pten^pc−/− and Pten^pc−/−;mTor^pc−/− 6 month old mice. (B) H&E staining of AP sections from 6 month old Pten^pc−/− and Pten^pc−/−;mTor^pc−/− mice. Focal invasion in Pten^pc−/− mouse prostate is indicated by an arrow. Inflammatory infiltration in Pten^pc−/− mouse prostate is shown at higher magnification in the lower left panel. Lower right panel: percentage of Pten^pc−/− and Pten^pc−/−;mTor^pc−/− 6 month old mice with signs of invasion in the prostate. Four mice from each genotyping were analyzed. **, P < 0.01.

(A) Western blot analysis of lysates of mTor^loxP/loxP, Pten^loxP/loxP and Pten^loxP/loxP;mTor^loxP/loxP MEFs infected with PURO-IRES-GFP (vector) or Cre-PURO-IRES-GFP (Cre) (see experimental timeline shown in Supplementary Fig. 3A). Quantification of the phospho-Akt/total Akt ratio is shown. Quantifications were done by densitometry analysis performed with ImageJ software. For each genotyping the phopho-Akt/total Akt value of Cre-infected cells is normalized against the corresponding vector-infected control. (B) Flow cytometric analysis (Forward Scatter, FSC-H) of the mTor-null (Pten^+/+;mTor^Δ/Δ-Cre) and Pten;mTor-double null MEFs (Ptenr^Δ/Δ;mTor^Δ/Δ-Cre) compared to the WT (Pten^+/+;mTor^+/+-Cre). (C) Cell proliferation curve analysis of the same MEFs analyzed in (B) followed over a 6-day period. (D) Cell proliferation curve analysis of mTor^loxP/loxP and Pten^loxP/loxP;mTor^loxP/loxP primary MEFs firstly immortalized with SV40 large-T antigen, and subsequently infected with PURO-IRES-GFP (vector) or Cre-PURO-IRES-GFP (Cre). (E) TUNEL assay on the same MEFs analyzed in (B). *, P < 0.05. (F) Flow cytometric analysis of mTor^loxP/loxP and Pten^loxP/loxP;mTor^loxP/loxP MEFs infected with PURO-IRES-GFP (vector) or Cre-PURO-IRES-GFP (Cre) (see experimental timeline shown in Supplementary Fig. 3A). In order to accurately evaluate cell cycle populations, data was gated to exclude the sub-G1 population. (G) Western blot analysis on the lysates of mTor^loxP/loxP and Pten^loxP/loxP;mTor^loxP/loxP MEFs infected with PURO-IRES-GFP (vector) or Cre-PURO-IRES-GFP (Cre) (see experimental timeline shown in Supplementary Fig. 3A).

(A) Representative images of prostates from 3 month old WT, Pten^pc−/− and Pten^pc−/−;mTor^pc−/− mice. (B) Upper panel: H&E staining of AP sections from 3 month old Pten^pc−/− and Pten^pc−/−;mTor^pc−/− mice. An example of residual PIN lesion in Pten^pc−/−;mTor^pc−/− mouse prostate is indicated by the arrow and shown at higher magnification. Lower panel: percentage of glands affected by PIN in the prostate of 3 month old Pten^pc−/− and Pten^pc−/−;mTor^pc−/− mice. Error bars show S.D. in the number of glands affected by PIN in 4 mice from each genotype. **, P < 0.01. (C) IHC staining of PIN lesions present in the prostates of Pten^pc−/− and Pten^pc−/−;mTor^pc−/− mice showed in (B upper panel) with anti-phospho S6 and anti-phospho S473 Akt antibodies. Quantification (+/− SD) of phospho-S6 and phospho-Akt positive cells in the PIN lesions of Pten^pc−/− and Pten^pc−/−;mTor^pc−/− mice is indicated. (D) Upper panel: H&E staining of AP sections from 8 weeks old Pten^pc−/− mice treated with vehicle or RAD001 for 4 weeks. One of the residual PIN lesions in RAD001-treated Pten^pc−/− mouse prostate is indicated by the arrow and shown at higher magnification. Lower panel: percentage of glands affected by PIN in the prostate of 8 weeks old Pten^pc−/− mice treated with vehicle or RAD001 for 4 weeks. Error bars show S.D. in the number of glands affected by PIN in 4 vehicle-treated and 4 RAD001-treated Pten^pc−/− mice. *, P < 0.05. (E) IHC staining of PIN lesions present in the prostate of the Pten^pc−/− mice treated with vehicle or RAD001 shown in D upper panel) with antibodies directed against phospho-S6 and phospho S473 Akt.

(A) Left panel: Ki-67 staining on prostate sections from 3 month old mice of the indicated genotypes. Right panel: quantification of Ki-67. Pos. stands for positive. Three different areas of one section were counted to determine an average and representative value for each slide. Slides from three independent mice were counted in this way to determine a standard deviation for the population. *, P < 0.05. The inset shows the Ki-67 quantification exclusively in the PIN lesions of the Pten^pc−/− and Pten^pc−/−;mTor^pc−/− mice prostates. (B) TUNEL assay for apoptosis on prostate sections from 3 month old mice of the indicated genotypes. The arrows show examples of TUNEL positive cells. The inset in the Pten^pc−/−;mTor^pc−/− prostate section shows the TUNEL staining in a residual PIN lesion. (C) Upper left panel: senescence-associated β-Galactosidase staining (SA β-Gal) on prostate sections from 3 month old mice of the indicated genotypes. Upper right panel: quantification of the SA β-Gal staining in the glands affected by PIN of the prostate sections from 3 month old mice of the indicated genotypes. Quantifications were done on three representative sections from three mice. There is not significant difference between the PIN lesions of Pten^pc−/− and Pten^pc−/−;mTor^pc−/− prostates. Pos. stands for positive. Lower panel: IHC for p53 on the same mice showed in the upper panel.