Additional file 1: of Alterations to mTORC1 signaling in the skeletal muscle differentially affect whole-body metabolism

Supplementary Figures S1 to S5. Figure S1. Unchanged expression of Actb. (A) Actb (encoding β-actin) expression in the muscle, liver, WAT, and BAT of control (n=5) and RAmKO mice (n=5). (B) List of antibodies used. Figure S2. Metabolism of female TSCmKO and RAmKO mice. (A) Body weight, (B) lean mass, (C) fat massand (D) plasma glucose levels of 10-week-old female TSCmKO (n=9), RAmKO (n=8) and control (Ctrl) mice (n=11). (E)–(G) Fat mass (E), lean mass (F) and plasma glucose levels (G) of male RAmKO (n=4) and control (Ctrl) mice (n=6) on a HFD. Figure S3. RAmKO mice do not show changes in other organs. (A) Glycogen amount in gastrocnemius muscle of 10-week-old TSCmKO mice (n=3). (B) Western blot analysis of liver from 10-week-old TSCmKO and control (Ctrl) mice (n=4). Mice were intraperitoneally injected with insulin (+; TSC-insulin) or not (−). Protein expression is normalized to eEF2. (C)–(D) 12-week-old RAmKO mice do not show changes in Ucp2 expression in the liver and WAT (C) or Ucp1 and Ucp2 in BAT (D) compared to control mice (n=5). (E)–(F) Liver expression of genes involved in lipid (E) and glucose (F) metabolism of RAmKO mice (n=5). Figure S4. ATP levels in 12-week-old RAmKO soleus muscle (n = 5). Figure S5. Body composition in myopathic female TSCmKO and RAmKO mice. (A) Body weight, (B) lean and (C) fat mass of 40-week-old TSCmKO (n=10), 20-week-old RAmKO (n=4) and respective control (Ctrl) littermates (n=11). (D) The kidneys of 40-week-old TSCmKO mice appear polycystic. Cysts are indicated by arrows

Additional file 2: of Alterations to mTORC1 signaling in the skeletal muscle differentially affect whole-body metabolism

Supplementary Tables S1 and S2. Table S1. List of primers used. Table S2. RAmKO blood analysis

Influence of SIRT3 depletion on protein expression of mitochondrial content markers.

<p>At the indicated states (Proliferation, P; Cell Confluence, C and 3 days after the induction of differentiation, D3), 50 µg of total protein from C2C12-LucshRNA (shCTL) and C2C12- SIRT3shRNA (shSIRT3) were immunoblotted with antibodies raised against PGC-1α, CS or Tubulin as an internal control. Results are expressed as the mean ± SD of three separate experiments. Quantification was performed with Image J software and normalized relatively to Tubulin protein levels. ANOVA main effect: ## P<0.01 <i>vs.</i> shCTL cells. Post-hoc significance: *P<0.05 <i>vs.</i> proliferating myoblasts for each cell type. §P<0.05 <i>vs.</i> shCTL cells at the same state.</p

MyoD overexpression in shSIRT3 restores myoblasts differentiation.

<p>C2C12 and C2C12-SIRT3shRNA (shSIRT3) cells were infected with pBabe empty vector and pBabe-MyoD vector retroviral supernatants. A) Immunostaining with an anti-Troponin T antibody 3 days after the induction of differentiation. Nuclei were stained with Hoechst. Microphotographs of a typical experiment are shown (x400). B) Representative Western-blot of Myogenin at cell confluence (C) and after 3 days of differentiation (D3).</p

Influence of SIRT3 depletion on mitochondrial activity.

<p>A) Respiration rates in C2C12- LucshRNA (shCTL) and C2C12-SIRT3shRNA (shSIRT3) at cell proliferation (P), cell confluence (C) and 3 days of differentiation (D3). Values are normalized relatively to total protein levels. B) Citrate synthase, complex II and cytochrome c oxidase maximal activities in shCTL and shSIRT3 cells at cell proliferation (P), cell confluence (C) and 3 days of differentiation (D3). Results are expressed as mean ± SD from five independent experiments. ANOVA main effect: #P<0.05, ##P<0.01 and ###P<0.001<i>vs.</i> shCTL cells. Post-hoc significance: *P<0.05, **P<0.01 and ***P<0.001 <i>vs.</i> proliferating myoblasts for each cell type. §P<0.05, §§P<0.01 and §§§ P<0.001 <i>vs.</i> shCTL cells at the same state.</p

SIRT3 depletion induces an oxidative stress.

<p>A) Intracellular ROS accumulation in C2C12- LucshRNA (shCTL) and C2C12-SIRT3shRNA (shSIRT3) at cell proliferation (P), cell confluence (C) and 3 days of differentiation (D3). Values are normalized relatively to total DNA levels. ANOVA main effect: ### P<0.001 <i>vs.</i> shCTL cells. Post-hoc significance: ***P<0.001 <i>vs.</i> proliferating myoblasts for each cell type. §P<0.05, §§§ P<0.001 <i>vs.</i> shCTL cells at the same state. B) MnSOD maximal activity in shCTL and shSIRT3 cells cell confluence (C). Results are expressed as mean ± SD from five independent experiments. *P<0.05 <i>vs.</i> shCTL cells.</p

Depletion of SIRT3 impairs terminal myoblast differentiation.

<p>Endogenous SIRT3 expression in C2C12-LucshRNA (shCTL) and C2C12-SIRT3shRNA (shSIRT3) cells during proliferation (P), at cell confluence (C) and after 3 days of differentiation (D3). A) SIRT3 mRNA expression was monitored by real-time RT-PCR and normalized relatively to the reference genes ARP and TBP. Results are expressed as the mean ± SD of three independent experiments. B) Western blot analysis of SIRT3 protein expression. Quantification was performed with Image J software and normalized relatively to Tubulin protein level. Results are expressed as the mean ± SD of three separate experiments. ANOVA main effect: ### P<0.001 <i>vs.</i> shCTL cells. Post-hoc significance: *P<0.05, **P<0.01 and ***P<0.001 <i>vs.</i> proliferating myoblasts for each cell type. §P<0.05, §§P<0.01 and §§§ P<0.001 <i>vs.</i> shCTL cells at the same statstatee. C) Immunostaining of C2C12, C2C12-LucshRNA (shCTL) and C2C12-SIRT3shRNA (shSIRT3) cells with an anti-Troponin T (differentiated state) or anti α-tubulin (undifferentiated state) antibody and fusion index 3 days after the induction of differentiation. Nuclei were stained with Hoechst. Microphotographs of a typical experiment are shown (x400). Fusion index values are expressed as the mean ± SD of 10 images/dish using image J software. *** P<0.001 <i>vs.</i> shCTL cells.</p

Depletion of SIRT3 impairs Myogenin, MyoD and SIRT1 expression.

<p>Western-blot analyses of Myogenin, MyoD, Sirt1 and Tubulin in C2C12-LucshRNA (shCTL) and C2C12-SIRT3shRNA (shSIRT3) cells at the indicated states (Proliferation, P; Cell Confluence, C and 3 days after the induction of differentiation D3). Results are normalized relatively to Tubulin protein levels and expressed as the mean ± SD of three separate experiments. ANOVA main effect: ## P<0.01, ### P<0.001 <i>vs.</i> shCTL cells. Post-hoc significance: *P<0.05, **P<0.01 and ***P<0.001 <i>vs.</i> proliferating myoblasts for each cell type. §P<0.05, §§P<0.01 and §§§ P<0.001 <i>vs.</i> shCTL cells at the same stage.</p