Fig 2: Western blot analysis of the mTOR network during osteoblastic differentiation of MSC. Cells were cultured for 21 days under control conditions (no OM) or with osteoblast induction medium in presence of vehicle (OM) or 20 nM Rapa (OM+Rapa). (A) Phosphorylation of p70-S6 kinase at threonine 389 (pp70-S6K389) for activation of mTORC1. (B) Phosphorylation of AKT at serine 473 (pAKT473) for activation of mTORC2. (C) Raptor as the key subunit of mTORC1. (D) Rictor as the key subunit of mTORC2. (E) mTOR as the serine/threonine kinase component of mTORC1 and mTORC2. (F) Phosphorylation of AKT at threonine 308 (pAKT308) as an upstream modulator and part of a feedback loop involving phosphatidyl inositol-3 phosphate kinase (PI3K). (G) Phosphorylation of ERK1/2 at threonine 202/tyrosine 204 (pERK202/204), and (H) its downstream target p70-S6 kinase at threonine 421/serine 424 (pp70-S6K421/424) as a side branch of p70-S6K activation. Densitometric quantifications and representative western blots are shown. Numbers of independent experiments are indicated at the bottom of each graph. Band intensities were normalized to GAPDH as a loading control. Control (no OM) was set to 1. Bars represent mean + SEM, *P < 0.05, **P < 0.01, ***P < 0.001. (I) Scheme of the mTOR network without, and (J) with modulation by Rapa.

Fig 3: mTORC1 inhibitors differentially affect the expression of BCL-2 family proteins(A) The mean log2 fold change in viable cell number, via trypan blue exclusion, of Tsc2-/- MEFs treated with vehicle (DMSO) or rapamycin (20 nM) for 72h in 0.5% serum is graphed as mean ± SD relative to Day 0. n = 2. *p<0.05 (two-tailed Student’s t test).(B) The percent of Tsc2-/- MEFs treated as in (A), but for 48 h. Annexin V/PI staining was quantified. n = 4.(C) Heat maps of RT-qPCR data from Tsc2-/- MEFs and Tsc2-/- 105K renal tumor cells treated with vehicle, rapamycin (20 nM), or Torin1 (250 nM) for 24h without serum. Heat maps represent the log2 transformation of transcript levels relative to the vehicle-treated samples (graphs shown in Figures S1A and S1B). n = 2.(D and E) Immunoblots of Tsc2+/+ and Tsc2-/- MEFs (D) and Tsc2-/- 105K cells reconstituted with TSC2 or empty vector (E) treated with vehicle (DMSO), rapamycin (20 nM), Torin1 (250 nM), AZD2014 (1 µM), or Rapalink-1 (5 nM) for 24 h without serum.(F–I) Immunoblot of Tsc2-/- MEFs (F,H) and Tsc2-/- 105K cells (G,I) treated as above with vehicle (0h time point), rapamycin (F,G), or Torin1 (H,I) for the indicated times.(J) Immunoblot of Rictor+/+ and Rictor-/- MEFs treated with vehicle (DMSO), or Torin1 (250 nM) for 24 h in full serum.

Fig 4: Oleic acid induces ACLY phosphorylation in an mTORC2 dependent manner.(A) HCT116 cells were plated at 60% confluency in complete medium (10% serum) and shifted to serum-free medium for 17 hr. OA (10 µM):BSA in a 2:1 ratio was added for 60 min. Fatty acid-free BSA was used as a negative control since OA was carried in complex with BSA. Lysates were collected and analyzed for phosphorylated Akt (S473) and ACLY (S455) via Western blot. (B) HCT116 cells were plated at 50% confluency in complete medium. Knockdown of Rictor was carried out via Rictor siRNA (100 nM) (+) or non-targeting (-) siRNA for 72 hr. At the 48 hr point of knock down, the media was changed to serum free media for overnight. OA (10 µM):BSA in a 2:1 ratio was added in presence or absence of Rictor. Insulin (100 nM) was also added to the HCT116 cells in the presence and absence of Rictor. Lysates were collected 60 min later and probed via Western blot for phosphorylated Akt and ACLY as in (A). (C) HCT116 cells were plated and treated as in A. Torin1 (1 µM) was used as an mTORC2 inhibitor. Lysates were collected and analyzed by Western blot. BSA, bovine serum albumin. The Western blots shown are representative of experiments repeated at least three times.

Fig 5: PTEN inhibits mTORC2 formation by hyperphosphorylation of Rictor at Threonine 1135 position. (a) PTENmu and PTENwt cells were lysed for co-immunoprecipitation (co-IP) study. Protein was incubated with anti-mTOR antibody overnight at 4oC followed by incubation with protein A-Sepharose 4B, washed with ice-cold phosphate-buffered saline (PBS). The immunocomplex was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and subsequently identified using the respective antibodies. Co-IP revealed that Raptor is associated with mTOR in both the cells, whereas Rictor is associated with mTOR only in PTENmu cells. (b) PTENwt cells were transfected with siPTEN. After 24 h of transfection, cell lysates were subjected to co-IP analysis. Cells transfected with siPTEN showed increased association of Rictor with mTOR. (c) Untreated PTENwt and PTENmu cell lysates were subjected for western blot analysis. Expression of phosphorylated Rictor at Thr1135 was detected higher in PTENwt than PTENmu cells. (d, e) PTENwt cells were transfected with siPTEN. After 24 h of transfection cell lysates were subjected to western blot analysis. Status of Rictor phosphorylation at Thr1135 position was determined. Densitometric analysis showed that there was about 35% reduction of phosphorylation of Rictor (Thr1135) after PTEN knockdown from PTENwt cells. Each value is the mean±s.d. of three independent experiments. *P<0.05, significant difference between two test groups. (f) Other PTENmu (T98G, U373MG) and PTENwt (LN18) GBM cell lysates were prepared and subjected for western blot analysis. Status of Rictor Thr1135 phosphorylation was compared. (g, h) PTENwt cells (8 × 105) were seeded and incubated for 24 h. Next, cells were transfected with wild-type pRK-5/Rictor (myc-tag) or the T1135A mutant of pRK-5/Rictor (myc-tag, 2 µg/well) or empty vector alone. After 36 h of transfection, cell lysates were prepared and subjected to co-IP (g) and western blot analysis (h). (g) Co-IP showed Rictor was associated with mTOR in cells transfected with pRK-5/Rictor T1135A. (h) Immunoblot analysis showed the status of myc-tagged Rictor, p-AKT Ser473 and p-GSK3ß Ser9 in those transfected cells. (i) Schematic representation: PTEN-mediated regulation of mTORC2 formation and downstream signaling. (j, k) PTENwt cells were transfected with siRaptor or co-transfected with siRaptor and siPTEN consecutively. After 24 h of transfection, cells were lysed and subjected to western blot analysis. (j) Western blot showed the relative status of mTOR, p-mTOR, p-S6K1, Rictor, p-Rictor Thr1135, AKT, p-AKT Ser473, Raptor and PTEN. (k) Densitometry analysis showing the fold change of p-mTOR Ser2481 and p-Rictor Thr1135. Each value is the mean±s.d. of three independent experiments. *P<0.05, significant difference between two test groups. In all the western blot experiments, ß-actin served as loading control.