Fig 2: mTORC1-mediated consequences on cell cycle and cell size are separable and do not involve effects on mTORC2 activity. (A) Logarithmically growing non-transformed, non-immortalized IMR-90 fibroblasts, derived from human fetal lung tissue, were treated with the mTOR inhibitor rapamycin at final concentrations ranging from 0.1 nm to 100 nm for 24 h. Control cells were treated with an equal volume of DMSO (vehicle). Total lysates were prepared and analysed for the expression level of p70S6K and Akt as well as for the phosphorylation status at T389 and S473, respectively. (B) In addition, so treated cells were stained with propidium iodide and cytofluorometrically analysed for DNA distribution. Representative DNA profiles are presented (upper panel). The percentage of cells in G0/G1, S and G2/M is indicated (lower panel). (C) Phospho-Akt S473 signals detected in (A) were densitometrically scanned and normalized to total Akt levels. (D) 100 nm rapamycin-treated IMR-90 cells derived from the same pool of cells as analysed in (A, B and C) were cytofluorometrically investigated for overall cell size via FSC (Forward Scatter) analyses. Overlays of FSC histograms are presented to enable direct comparison of control cells versus rapamycin-treated cells (left panel). Mean FSC values obtained from these analyses are presented (right panel). (E) In addition, cell size according to different cell cycle phases was examined via two-dimensional contour blot analyses of FSC versus DNA content. To visualize FSC shifts between different samples, a crosshair has been set into the center of the G0/G1 population of control cells (left panel). For details regarding different types of FSC analyses compare the Methods section in the text. Furthermore, G0/G1-phase cells (2 N) and G2/M-phase cells (4 N) were selectively gated and analysed for cell size distribution via FSC. Mean FSC values and the percentage of gated cells are presented (right panel). (F) Lysates of IMR-90 cells treated with 100 nm rapamycin for the indicated times were analysed for the expression level of total p70S6K (on long exposures the used a-p70S6K antibody recognizes also lower migrating p85S6K) and total Akt as well as for their phosphorylated forms as indicated. In addition, same lysates were examined for cyclin A levels. (G) Logarithmically growing IMR-90 cells (vehicle) were either treated with 100 nm rapamycin for 24 h as described in (A) or were grown under low proliferation conditions. So treated cells were cytofluorometrically analysed for DNA distribution. The percentage of cells in S-phase is indicated. (H) In parallel, overall cell size was cytofluorometrically investigated via FSC (upper panel). Mean FSC values obtained from these analyses are presented (lower panel). (I) In addition, cell size according to different cell cycle phases was examined via two-dimensional contour blot analyses of FSC versus DNA content. (J) IMR-90 fibroblasts were treated with 100 nm rapamycin for the indicated times and total cell lysates were analysed for the expression levels of p70S6K T389, total p70S6K, Akt S473, total Akt, cyclin A and cyclin D1. In addition, the percentage of cells in S-phase was determined on the flow cytometer. (K) Phospho-Akt S473 signals detected in (J) were densitometrically scanned and normalized to total Akt levels. (L) In parallel, overall cell size distribution of so treated cells was cytofluorometrically analysed via FSC at the indicated time points. (M) Logarithmically growing IMR-90 fibroblasts were transfected with pooled short-interfering RNAs (siRNAs) targeting human rictor (si rictor). Cells treated with non-targeting siRNAs (control) or cells which were left entirely untreated were analysed in parallel. About 72 h after initial transfection, lysates were prepared and analysed for indicated proteins via immunoblotting. (N) Experiments were basically performed as described in (M) with the exception that cells were treated with 100 nm rapamycin or DMSO for the final 24 h of incubation. So treated cells were lysed and examined for the expression level of rictor, S473 phosphorylated Akt, total Akt, T389 phosphorylated p70S6K and total p70S6K.

Fig 3: mTORC1 is activated in obese mouse livers, and is involved in nutrient-repletion-induced down-regulation of CDK8 in tissue culture.Control wild-type and db/db (A) or ob/ob (B) male mice on the C57Bl/6J background were maintained on the normal chow diet. At 4 months of age, the mice were fasted for 16 hours and then re-fed for 4 hours before sacrifice. Liver extracts were prepared and analyzed for the levels of S6K1 phosphorylation on T389 by immunoblotting. Total amount of S6K1 served as the loading control. The relative protein levels in the livers of db/db (C) or ob/ob (D) mice were further analyzed by densitometry. *p<0.05 and **<0.01 vs. wild-type (n = 3 independent mice). E) Effects of nutrient-depletion and nutrient-repletion on the levels of indicated proteins in the presence of mTOR inhibitor rapamycin or Torin 1. FAO cells were cultured in the low-serum DMEM overnight, and then in amino acid-free RPMI for 2 hour. The cells were pre-treated without or with 100nM rapamycin or 250nM Torin 1 for 30 min followed by the addition of regular DMEM containing 100nM insulin for the indicated period of time. Cell extracts were prepared and analyzed for the indicated proteins by immunoblotting. These are representative immunoblots independently performed for four times.

Fig 4: Schematic diagram showing the proposed mechanism underlying IL10-mediated inhibition of autophagy in starvation-treated HSFs. IL10 inhibits starvation-induced autophagy via IL10R-mediated activation of the IL10R-STAT3 pathway (IL10/IL10R-STAT3 pathway) or via direct activation of AKT-mTOR pathway (IL10/AKT-mTOR pathway). IL10 inhibits starvation-induced autophagy by inducing cross talk among STAT3, AKT, and mTOR, particularly STAT3 and mTOR and, finally, via activation of p70S6K ('?' activation, '?' inhibition)

Fig 5: Arg404/Asp406/Asp407 residues in Sesn-C constitute a functional site for mTORC1 regulation.(a) The surface of Sesn-C was subdivided into five different areas (highlighted in blue, purple, yellow, green and red) that contain the most highly conserved surface residues. In addition, the residues corresponding to the formerly described putative GDI motif (Arg419, Lys422 and Lys426)17 are highlighted in brown. These residues were mutated into alanines as described in Supplementary Table S1. (b,c) The R404A/D406A/D407A mutation (highlighted in yellow), but none of the other mutations, abolished the mTORC1-inhibiting activity of hSesn2. mTORC1-dependent phosphorylation of S6K was monitored by electromobility retardation of HA–S6K (shifted bands)26. HEK293 cells were transfected with plasmid constructs expressing HA-tagged S6K1 and Flag-tagged hSesn2 of the indicated mutations. GFP or an empty vector (S6K1 only) was used as negative controls. After 48 h of transfection, cell lysates were analysed by immunoblotting of the indicated proteins. (d) RKO cells were infected with lentiviral constructs expressing GFP or Flag-tagged hSesn2 of indicated mutations. After 48 h of infection, cell lysates were analysed by immunoblotting of the indicated proteins. Rapamycin (rap, 100 nM for 24 h) was used as a positive control for mTORC1 inhibition. Molecular weight markers are indicated in kDa.