Fig 2: LysM-Rictor KO mice exhibit a higher susceptibility to LPS-induced septic shock.LysM-Rictor WT and LysM-Rictor KOmice were injected intraperitoneally with LPS (2.5 mg/kg of body weight) and body temperature was measured 4 hours post-injection. * denotes a significant difference between LysM-Rictor WT or LysM-Rictor KO mice (p<0.05). Data are expressed as the mean ± SEM for n = 6–10 per condition. (B) Survival rate of LysM-Rictor WT or LysM-Rictor KO mice following the injection of LPS. Mice having a core body temperature dropping down to 33.0+/-0.2°C or below following LPS injection were killed. (C) The sensitivity of LysM-Rictor KO mice to LPS-induced hypothermia is linked to an elevation in circulating levels of TNF-a. Plasma was collected from the mice used in the experimentation described in A before LPS injection and 1,2, and 4 hours post injection. TNF-a levels were measured by ELISA. Data are expressed as the mean ± SEM. The graph presented in B is based on the plasma measurements of TNF-a of all LysM-Rictor WT mice (n = 6) vs. the LysM-Rictor KO mice that died over the experiment (n = 7). *p<0.05 versus control.

Fig 3: mTORC1 and mTORC2 directly phosphorylate p53 at S15 in PICS. a MCF7 cells exogenously expressing flag-tagged WT, ?40, and ?133 p53 along with Myc-tagged mTOR were transfected with Con Si or PTEN Si. Two days after siRNA transfection, the cells were immunoprecipitated with anti-Flag antibody. b Cells were transfected with Con Si or PTEN Si, harvested at the indicated days, and subjected to immunoblotting. c In vitro kinase assay. MCF7 cells were transfected with Con Si or PTEN Si. After 2 days, cell lysates were immunoprecipitated with a-Raptor and a-Rictor antibodies and used for in vitro kinase assay. d MCF7 cells exogenously expressing Flag-tagged WT p53 or S15A mutant p53, in combination with Myc-tagged mTOR, were transfected with Con Si or PTEN Si. Two days after siRNA transfection, the cells were immunoprecipitated with anti-Flag antibody. SA-ß-Gal activity assays were conducted 4 days after siRNA transfection. The values represent the mean ± SD; n = 3; #P > 0.05; ***P < 0.001

Fig 4: mTORC2 signaling plays no role in the development of vaginal epithelium.A The breeding strategy used to generate Rictor cKO female mice. B Expression of Rictor was quantified using qPCR in the vagina of control (n = 6) and Rictor cKO (n = 5) mice. C Rictor protein levels in control (n = 4) and Rictor cKO (n = 4) vagina tissues were determined by immunoblotting. ß-Actin was used as the loading control. The experiments were repeated two times. D Gross morphology of external vaginal orifice and reproductive tracts of control (n = 5) and Rictor cKO (n = 5) mice. E 3D renderings of the female reproductive tract in control (n = 4) and Rictor cKO (n = 4) mice based on MRI T2 weighted images. Scale bars: 5 mm. F Accurate max and min diameters of the vagina were measured based on E. Values are expressed as the mean ± SEM. G Representative images for cytological assessment of vaginal smears of control and Rictor cKO mice using crystal violet staining method for estrous cycle determination. n = 5 mice in each group. Microscopy with magnification ×20. Scale bars: 100 µm. H Representative HE-stained vaginal sections of OVX control and Rictor cKO mice are shown. Nuclei were stained with hematoxylin. n = 3 mice in each group. The experiments were repeated three times. Microscopy with magnification ×20. Scale bars: 50 µm.

Fig 5: Inducible depletion of RICTOR impairs male lifespan. (A) Kaplan–Meier plot showing survival of male wild-type and UbC-RKO mice treated with tamoxifen at 10 weeks of age. The survival of 26 mice was analyzed as follows: Males (11 wild-type, 15 UbC-RKO). (B) Kaplan–Meier plot showing survival of male wild-type and UbC-RKO mice treated with tamoxifen at 9 months of age. The survival of 25 mice was analyzed as follows: Males (10 wild-type, 15 UbC-RKO). Raw data and statistical information can be found in Tables S1 and S2. (C) Depletion of RICTOR results in gray hair and kyphosis. Pictured: 15-month-old wild-type (left) and UbC-RKO mice (right) from which Rictor was excised at 10 weeks of age. (D) Weight change of wild-type and UbC-RKO mice over 30 weeks, starting at 10 weeks of age.