Nardella et al.

Supplementary Fig. 1 Characterization of PB-Rheb transgenic mice. (A) PCR analysis of genomic DNA from founder animals. (B)Quantitative RT-PCR analysis of the prostate RNA from PB-Rheb mice with a murine Rheb specific probe. Error bars show S.D. from three independent experiments.(C) Immunoprecipitation on protein lysates from WT and PB-Rheb transgenic mice prostate with anti-Rheb antibody and probed with anti-HA antibody. Control: immunoprecipitation on protein lysate from HeLa cells tranfected with pcDNA3.1 HA-Rheb.

Supplementary Fig. 2 Rheb-overexpression-induced senescence in vitro. Upper panel: western blot analysis of primary mouse embryonic fibroblasts (MEFs) infected with HA-Rheb-PURO-IRES-GFP (HA-Rheb-PIG) or control retrovirus (PIG). Center and lower panel: SA -Gal staining for senescence and its quantification of MEFs infected with PIG or HA-Rheb-PIG) treated with vehicle or rapamycin. Error bars show S.D.

Supplementary Fig. 3 Characterization of PB-Rheb;Pten+/-micey. (A) Pten (left panel) and phospho-Akt (right panel) staining of prostate sections from 10-month old Pten+/- and PB-Rheb; Pten+/- mice. (B)Western blot analysis of protein extracts from AP of 5-month old mice of the indicated genotype. (C) Quantification of SA -Gal staining on prostate sections of 6-month old mice from the indicated genotypes. Quantifications were done on three representative sections from three mice for each genotype.

Fig.1 Rheb expression in human prostate cell lines and generation of PB-Rheb mouse model. (a) Western blot analysis of Rheb in a series of human prostate cell lines. Error bars show S.D. from three independent experiments. (b) Upper panel: schematic representation of the transgene construct. Center panel: Southern blot analysis of genomic DNA from potential founder animals. Lower panel: PCR analysis on the same DNA samples in the center panel. The expected size of the amplified product is approximately 200 bp. (c) Quantitative RT-PCR analysis of the prostate RNA from PB-Rheb mice with a human Rheb-specific probe. Error bars show S.D. from three independent experiments. (d) IHC staining of prostate sections from the WT and the two PB-Rheb lines with anti-Phospho S6.

Fig. 2 Histopathological analysis of the PB-Rheb mice prostates. (a) Upper panel: incidence of hyperplasia in the dorsolateral prostate (DLP) of the mice from the two different PB-Rheb transgenic lines compared with WT. Lower panel: DLP from WT and PB-Rheb were stained with hematoxylin and eosin (H&E). (b) Upper panel: incidence of low-grade PIN in the DLP of the mice form the two different PB-Rheb transgenic lines. Lower panel: DLP from WT and PB-Rheb were stained hematoxylin and eosin (H&E). Nuclear athypia has been indicated by arrows.

Fig. 3 Inverse association between hyperplasia and cellular senescence in the different lobes of the PB-Rheb mice prostates. (a) Senescence associate -Galactosidase staining (SA -Gal) in the three different lobes DLP, anterior prostate (AP) and ventral prostate (VP) from WT and PB-Rheb mice. (b) Left panel: incidence of hyperplasia in the different lobes of the prostates from 6-8 months old PB-Rheb mice. Right panel: quantification of senescence in the different lobes of the prostates from 6-8 months old PB-Rheb mice measured by (SA -Gal) staining as described….

Fig. 4 Cooperativity between Rheb overexpresison and Pten heterozygous loss in vivo. (a) Incidence of high grade PIN in the AP of five 10 months old mice of each genotype. (b) Histopathological (H&E) analysis of the AP from mice of each genotype (c) IHC staining of 5-months old mice of each genotype with anti-Phospho S6. (d) Western blot analysis of protein extracts from AP of 5 months mice of the indicated genotype. (e) Ki-67 staining in the prostate from 6 months old mice of the indicated genotypes. Bottom panel panel: quantification of Ki-67. Comment in both SD….

Supplementary Fig. 1 Mouse Rheb expression does not change in the prostate of PB-Rheb mice. Quantitative RT-PCR analysis of the prostate RNA from PB-Rheb mice with a murine Rheb-specific probe. Error bars show S.D. from three independent experiments.

Supplementary Fig. 2 Rheb-induced senescence in vitro (a) Upper panel: western blot analysis of primary mouse embryonic fibroblasts (MEFs) infected with HA-Rheb-PURO-IRES-GFP (HA-Rheb-PIG) or control retrovirus (PIG). Center and lower panel: -Gal staining for senescence and its quantification of MEFs infected with PIG or HA-Rheb-PIG) treated with vehicle or rapamycin. Comment SD

Supplementary Fig. 3 Status of the AKT-mTOR pathway components in neoplastic lesions and senescence in vivo in the context of Rheb overexpression and Pten heterozygisity. (a) PTEN and (b) Phospho-Akt staining of ptostates from 10-months old Pten+/- and PB-Rheb;Pten+/- mice. Left panel: SA -Gal staining in the prostates of 6 months old mice from the indicated genotypes. Right panel: quantification of senescence in the prostate of 6 months old mice from the indicated genotypes. Comment SD

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