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 α-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.

Blocking mTORC1 activity causes G1 cells to enter S-phase at smaller size. IMR-90 fibroblasts, synchronized and restimulated in the presence or absence of 100 nm rapamycin as described in Figure 2, were analysed on a flowcytometer. At specific time points as indicated, cells from the experiment presented in Figure 2B and C were examined for overall cell size via FSC analyses (A) (in addition, mean FSC values for each time point are presented in numbers) and for cell size according to different cell cycle phases via two-dimensional analyses of FSC versus DNA content (B) (in addition, the percentage of cells in S-phase is presented for each time point). (C) Cells from the experiment presented in Figure 2B and C were analysed in more detail. DMSO-treated cells (vehicle) at 15 h restimulation were compared with rapamycin-treated cells at 27 h restimulation via two-dimensional FSC analysis (left panel). In addition, G0/G1-phase cells and S+G2/M-phase cells were selectively gated and analysed for cell size distribution via FSC (upper right panel). Mean FSC values are presented (lower right panel). (D) Rapamycin or vehicle-treated cells derived from the same experiment presented in Figure 2D and E were analysed as outlined in (C) with the exception that DMSO-treated cells (vehicle) at 15 h restimulation were compared with rapamycin-treated cells at 24 h restimulation.

mTORC2 regulates cell size and cell cycle via a mechanism involving TSC2. (A) Simplified schematic presentation of the potential functional interaction of mTORC2 and mTORC1 in cell size regulation. Both, PI3K and mTORC2 (mTOR/rictor) are necessary to drive full activation of the serine-threonine kinase Akt, which in turn negatively regulates PRAS40 and the TSC1/2 complex to activate mTORC1 (mTOR/raptor) known to exert positive effects on cell size regulation via its effector S6K. (B–F) Logarithmically growing IMR-90 fibroblasts were either transfected with rictor siRNA alone or were co-transfected with siRNAs targeting rictor and PRAS40 or rictor and TSC2. Non-targeting siRNA (control) was analysed in parallel. Sixty hours post transfection, cells were replated into fresh growth medium and incubated for additional 20 h. For experiments presented in (D), cells were transfected as described above but were grown for a total of 96 h without replating. (B) siRNA-treated cells were harvested, lysed and protein levels of rictor, PRAS40 and tuberin were examined via immunoblotting to prove efficient knockdown of target proteins. In addition, the expression level of Akt and p70S6K as well as the corresponding phosphorylation status at S473 and T389, respectively, were analysed. Lysates of non-treated cells, which were co-analysed in parallel, were included to prove the here used conditions of siRNA treatment to be physiologic and technically sound (compare control versus non-treated). (C) siRNA-treated cells derived from the same pool of cells described in (B) were stained with propidium iodide and cytofluorometrically analysed for cell cycle distribution. Representative DNA profiles (upper panel) and the percentage of cells in G0/G1, S and G2/M (lower panel) are presented. (D) Lysates of cells treated as indicated were examined for the expression levels of rictor, tuberin, p27 and α-tubulin. In addition, cells were examined for overall cell size via FSC analyses (E) and for cell size according to different cell cycle phases via two-dimensional blots of FSC versus DNA-content (F) on the flow cytometer.

Blocking mTORC1 activity delays S-phase induction without effects on mTORC2 activity. IMR-90 fibroblasts, synchronized in G0/G1 via serum deprivation, were serum restimulated to re-enter the cell cycle in the presence or absence of the mTOR inhibitor rapamycin. (A) Schematic outline of the synchronization procedure. Briefly, cells growing under full serum (10% FCS) conditions for 24–72 h were serum-deprived in medium containing 0.2% serum (0.2% FCS) for 48 h and serum restimulated (10% FCS) for additional 36 h. Rapamycin was added 30 min prior to restimulation at a concentration of 100 nm. Control cells were treated with an equal volume of DMSO and analysed in parallel. (B) Cells treated as described in (A) were lysed and examined for the expression level of cyclin D1, cyclin A, p70S6K T389, p70S6K, Akt S473 and Akt at the indicated time points via immunoblotting. (C) In addition, cells were cytofluorometrically analysed for DNA content at the indicated time points. DNA profiles are presented (upper panel). In addition, the percentage of cells in S-phase was determined for DMSO (vehicle)- and rapamycin-treated cells. To facilitate interpretation of S-phase induction, a baseline corresponding to the S-phase values of serum-deprived vehicle- and rapamycin-treated cells was inserted into the graph. (D) Experiments as described in (A, B and C) were repeated and the percentage of cells actively undergoing DNA replication was analysed by immunocytochemical BrdU incorporation assay at specific time points as indicated (at least 250 cells were scored for each timepoint). (E) In addition, cells derived from the same pool of cells as described in (D) were immunocytocemically stained for cyclin A expression (at least 250 cells were scored for each timepoint).

mTORC2 regulates mammalian cell size and cell cycle progression. Logarithmically growing IMR-90 fibroblasts were transfected with short-interfering RNAs (siRNAs) targeting human raptor and rictor, respectively. Cells treated with non-targeting siRNA were analysed in parallel and served as a negative control (control). Forty-eight hours after transfection, cells were replated at low density and were grown for another 20 h. (A) Cells as described above were lysed and examined for the expression level of raptor, rictor, S473 phosphorylated Akt, total Akt, T389 phosphorylated p70S6K and total p70S6K. α-Tubulin was co-analysed as an additional loading control. (B) Cells derived from the same pool of cells described in (A) were cytofluorometrically analysed for DNA distribution. Representative DNA profiles (upper panel) and the percentage of cells in G0/G1, S and G2/M (lower panel) are presented. Apart from the quantification of DNA distribution, cells were cytofluorometrically examined for overall cell size via FSC analyses (C) and for cell size according to different cell cycle phases via two-dimensional blots of FSC versus DNA content (D).

The role of Rheb and Akt for mTORC2-mediated cell size effects. (A) IMR-90 cells were transfected with siRNAs targeting either TSC2, Rheb or both. Lysates of so treated cells were examined for the expression levels of tuberin, phospho-p70S6K T389 and total p70S6K. A non-specific band (NB) serves as a loading control. (B–D) IMR-90 fibroblasts were either treated with rictor siRNA alone or were co-transfected with siRNAs targeting rictor and TSC2 or rictor, TSC2 and Rheb, respectively. Cells were replated into fresh growth medium 60 h post transfection and incubated for additional 20 h. (B) So treated cells were stained with propidium iodide and cytofluorometrically analysed for overall cell size via FSC analyses. (C) Mean FSC values obtained from these analyses are presented. (D) In addition, cell size according to different cell cycle phases was examined via two-dimensional blots of FSC versus DNA-content. (E–H) Rictor- and control siRNA-treated cells were grown for 72 h, replated and transiently transfected with an empty expression vector or with an expression vector containing myristoylated (constitutively active) Akt (myrAkt) together with GFP-spectrin expression plasmids as reporter. Another 36 h later, cells were harvested and analysed via immunoblotting and flowcytometry. (E) The expression levels of rictor, ectopic Akt (via myc-antibody) and α-tubulin were analysed. (F) In addition, GFP-positive cells were examined for overall cell size via FSC analyses. (G) Corresponding mean FSC values obtained from these analyses are indicated. (H) Cell size according to different cell cycle phases was examined via two-dimensional blots of FSC versus DNA content.