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

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

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

Influence of chicoric acid on AMPK/ACC pathway in L6 myotubes.

<p>Cells were treated with the indicated concentrations of CA for 1(Thr172) and tubulin expression (<b>A</b>); influence of compound C on AMPK phosphorylation (<b>B</b>); phosphorylated ACC (Ser79) and tubulin expression (<b>C</b>). Data are expressed relatively to control value (without CA). Values are means ± SEM (n = 5). Significantly different from control at * p<0.05, ** p<0.01. Representative immunoblots are shown.</p

Influence of chicoric acid on Akt and mTOR phosphorylations in L6 myotubes.

<p>Cells were treated with 5 or 50 µM CA for 1 hour with or without 100 nM insulin and proteins were extracted for western blot analyses of phosphorylated Akt (Ser473) or tubulin (<b>A</b>), phosphorylated mTOR (Ser 2448) or tubulin (<b>B</b>). Data are expressed relatively to the value obtained with insulin (without CA) (<b>A</b>) or relatively to the value obtained without insulin (without CA) (<b>B</b>). Values are means ± SEM (n = 5). * p<0.05, ** p<0.01. Representative immunoblots are shown.</p

Chicoric acid-dependent lifespan modulation of <i>C. elegans</i>.

<p>The tabulated data show the average results plotted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078788#pone-0078788-g008" target="_blank">Figure 8</a>. Up to two independent trials were done at 20°C with identical results. Worms were fed with the E. coli HT115 strain bacteria (see Material and Methods section). XLSTAT-life statistical software (Addinsoft, New York, NY, USA) was used to plot survival data by the Kaplan Meier method and differences between survival curves calculated using the Log-Rank test with 95% confidence. (a) Represents the 50th percentile (the age when the survival fraction of animals reaches 0.50). (b) Experiment identification code. (c) Probability of being identical to other lifespan experiments given in parentheses. (d) Total death scored (number of censored values).</p

Influence of chicoric acid on mitochondrial activity in L6 myotubes.

<p>Cells were cultured in the presence or absence of 50 µM CA for 10 days before measurement of mitochondrial activities. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078788#pone-0078788-g004" target="_blank">Figure 4</a> illustrates complex II maximal activity (CII; <b>A</b>); complexes II+III maximal activity (CII+III; <b>B</b>); complex IV maximal activity (CIV; <b>C</b>); and citrate synthase maximal activity (CS; <b>D</b>). Enzymatic activities are expressed as mU/mg protein and indicated as means ± SEM (n = 5); significantly different from control at * p<0.05.</p