(A) Serial sections from wild-type, Pten^LoxP/LoxP PB-Cre⁺ or Pten^LoxP/LoxP Rictor^LoxP/LoxP PB-Cre⁺ tissue stained by H&E, or labeled with antibodies to phospho-Akt^S473 or to FoxO1 are shown. The dotted circles indicate phospho-Akt^S473 positive cells in which Foxo1 is excluded from the nucleus. Arrows point to phospho-Akt^S473 negative cells in which Foxo1 concentrates in the nucleus. An enlarged section is show in the boxed insert. Scale bar = 25μm. (B) Serial sections from wild-type, Pten^LoxP/LoxP PB-Cre⁺ or Pten^LoxP/LoxP Rictor^LoxP/LoxP PB-Cre⁺ tissue labeled with antibodies to phospho-Akt^S473, phospho-GSK3β^S9, or phospho-S6^S235/236 are shown. Arrows indicate regions highlighted in the boxed inserts. The arrowhead points to invasive cells. The encircled area in the right panels indicates a patch of phospho-Akt^S473 positive cells. Scale bar = 25μm.

(A) Prostate tissue serial sections from 9-week old mice were stained by H&E or labeled with a phospho-Akt^S473 antibody and imaged at 10X. The arrows point to changes in the stroma. The arrowhead indicates a patch of phospho-Akt^S473 positive cells. Scale bar = 50μm. (B) Higher magnification images (20X) of serial sections stained by H&E or labeled with a phospho-Akt^S473 antibody. Invasive phospho-Akt^S473 positive cells are indicated with an arrow. Scale bar = 50μM. (C) Labeling with Ki-67 antibody (left), trichrome stain (middle) and Rictor antibody (right) are shown. The arrow indicates proliferating (Ki-67 positive) cells in the stroma. The arrowhead points to a small patch of cells that have not lost Rictor expression. Scale bar = 50μm.

(A) Generation of PC-3 cells with stable knockdown of Rictor. Using a lentiviral delivery system, two independent shRNAs (shRictor¹ or shRictor²) that silence Rictor expression or a control shRNA targeting luciferase (shLuc) were stably expressed in PC-3 cells. Protein lysates prepared 6 days post-infection were probed with the indicated antibodies. (B) Rictor knockdown impairs cell proliferation in vitro. PC-3 cells stably expressing shLuc, shRictor¹ or shRictor² were seeded in triplicate at equal density and cell number was counted on four consecutive days. Fold change in cell number is shown. (C) PC-3 cells require mTORC2 to form tumors as xenogfrafts. PC-3 cells stably expressing shLuc, shRictor¹ or shRictor² were injected subcutaneously into nude mice. Tumors were dissected and photographed 28 days post injection. Scale bar = 2mm (D) Average tumor volume (mm³) and SEM is plotted.

(A) Deleting Rictor in combination with Pten blocks hyper-phosphoyrlation of Akt^S473 in MEFs. Pten^LoxP/LoxP or Pten^LoxP/LoxP Rictor^LoxP/LoxP MEFs were infected with Adeno-Cre virus. After 5-days, cells were starved and stimulated with 300nm insulin for 15 minutes. Protein lysates were prepared and probed with the indicated antibodies. For the Rictor immunoblot, ‘”long” indicates longer exposure. (B) The PB-Cre4 transgene induces recombination of Pten and Rictor conditional alleles only in prostate tissue and not in bladder or tail tissue. A sample set of PCR reactions is shown. DLV indicates pooled tissue from dorsal, lateral, and ventral prostate. (C) Deleting Rictor in combination with Pten inhibits Akt^S473 phosphorylation and activity towards downstream substrates in vivo. Protein lysates were prepared from dorsolateral prostates dissected from wild-type, Pten^LoxP/LoxP PB-Cre⁺ or Pten^LoxP/LoxP Rictor^LoxP/LoxP PB-Cre⁺ mice and probed with the indicated antibodies. (D-E) Rictor deletion does not affect normal prostate architecture but is required for Akt^S473 phosphorylation induced by Pten deletion. (D) Serial sections of 7-week old prostate tissue stained by H&E, or labeled with antibodies to Rictor or phospho-Akt^S473 are shown. Arrows point to a patch of cells in which inefficient Rictor deletion results in abnormal Akt^S473 positive cells. Scale bar = 50μM. (E) Cells lacking PTEN and phospho-Akt^S473 appear normal (circled area), consistent with loss of mTORC2 activity blocking transformation. For comparison, this example includes a nearby section of hyperplastic cells (indicated by the star) that stain negative for PTEN and positive for Akt^S473, which is indicative of inefficient Rictor deletion. Scale bar = 50μm.

(A) Pten^+/- mice lacking one mtor, mlst8, or Rictor gene tend to live longer. Survivability was monitored for 1 year and displayed using Kaplan-Meyer plots. (B-C) Rictor^+/- MEFs have reduced Akt activity. (B) Akt was isolated from serum deprived (starved) or stimulated (+FBS) MEFs that were wild-type or deleted for one or both Rictor genes. The ability of immunopurified Akt to incorporate radiolabeled phosphate into a synthetic substrate was measured. Bars indicate standard error. (C) Immunoblot corresponding to (b) showing phospho-Akt^S473 and total Akt levels. (D) Adeno-Cre was added to MEFs harboring conditional alleles of Pten and Rictor (Pten^L/L and Pten^L/LRictor^L/+) to generate Pten-null MEFs lacking only one Rictor gene. Empty vector was added to generate control cells. Lysates were prepared from serum-deprived cells that were stimulated with 0, 1, 10, or 100nm insulin for 10 minutes and probed with the indicated antibodies. (E-F) Pten^+/-Rictor^+/- mice are protected against prostate tumor development. (E) GU tracts of wild-type, Pten^+/- and Pten^+/-Rictor^+/- mice that survived to 1 year were dissected and photographed. Bladder (B) and Anterior Prostate (AP) are indicated for orientation. An example of a large tumor associated with a Pten^+/- prostate is circled with a white dotted line. (F) Pten^+/- and Pten^+/-Rictor^+/- prostate tissue was sectioned and stained by Hematoxylin & Eosin (top) or labeled with Ki-67 antibody (bottom). Representative images are shown. The star (*) indicates a normal prostate ductule and the arrow points to a ductule containing large hyperplastic cells. Scale bar = 50μm (top) and 25μm (bottom).