Involvement of SPARC in myoblasts fusion.

<p>Myoblasts (4×10⁴ cells/well in 24-well plates) were induced to differentiate in DM with/without the induction/inhibition of SPARC for 5 days. The mediums were changed each 48 h. C2C12 were stained with May-Grünwald and Giemsa solutions, and the stained wells were observed under light microscopy. One-way ANOVA followed by the Tukey's HSD post-hoc test was used. All results are reported as means ± SEM. The addition of rSPARC (2 μg/ml) induced myotube formation, whereas, anti-SPARC (10 μg/ml) inhibited it. (a) Captured microscopic images of C2C12 fusion. The images were captured using 30× objectives under light microscopy (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192714#pone.0192714.s002" target="_blank">S2 Fig</a> for 100× objectives). Examples of multinucleated myotubes were shown by arrows. (b) Fusion index was calculated from 10 different photos per condition. Abbreviations: PBS: phosphate buffered saline, rSPARC: recombinant SPARC protein, and anti-SPARC: anti-SPARC antibody. *Significant differences between experimental conditions (n = 3): p < 0.05.</p

SPARC effect on ECM proteins in differentiating myoblasts.

<p>C2C12 cells were cultured in DM for 5 days with/without exogenous rSPARC (2 μg/ml) and/or anti-SPARC (10 μg/ml). The mediums were changed each 2 days. Proteins extraction was performed, and western blot analysis was done on RIPA soluble lysates to indicate proteins. The experiments were repeated three times (three different passages of C2C12 cells) and a representative western blot image was shown. Data were expressed as a ratio to the positive control (pooled samples). One-way ANOVA with repeated measurements was used to adjust the validations of the different passages and a contrast analysis was performed. P value was set at < 0.05 after the Bonfferoni adjustments. (a): collagen 1a1 levels were measured using the 30 μg proteins and the results showed that the induction of SPARC increased collagen 1a1 expression, however, an opposite effect of anti-SPARC was observed. (b) Five μg of the proteins were loaded to measure fibronectin levels. A decrease of fibronectin expression with rSPARC and an increase after anti-SPARC were observed. Abbreviations: PBS: phosphate buffered saline, rSPARC: recombinant SPARC protein, and anti-SPARC: anti-SPARC antibody. *Significant differences between experimental conditions.</p

Implication of SPARC in the modulation of the extracellular matrix and mitochondrial function in muscle cells

<div><p>Secreted protein, acidic and rich in cysteine (SPARC) is differentially associated with cell proliferation and extracellular matrix (ECM) assembly. We show here the effect of exogenous SPARC inhibition/induction on ECM and mitochondrial proteins expression and on the differentiation of C2C12 cells. The cells were cultured in growth medium (GM) supplemented with different experimental conditions. The differentiation of myoblasts was studied for 5 days, the expressions of ECM and mitochondrial proteins were measured and the formation of the myotubes was quantified after exogenous induction/inhibition of SPARC. The results indicate that the addition of recombinant SPARC protein (rSPARC) in cell culture medium increased the differentiation of C2C12 myoblasts and myogenin expression during the myotube formation. However, the treatment with antibody specific for SPARC (anti-SPARC) prevented the differentiation and decreased myogenin expression. The induction of SPARC in the proliferating and differentiating C2C12 cells increased collagen 1a1 protein expression, whereas the inhibition decreased it. The effects on fibronectin protein expression were opposite. Furthermore, the addition of rSPARC in C2C12 myoblast increased the expression of mitochondrial proteins, ubiquinol-cytochrome c reductase core protein II (UQCRC2) and succinate dehydrogenase iron-sulfur subunit (SDHB), whereas the anti-SPARC decreased them. During the differentiation, only the anti-SPARC had the effects on mitochondrial proteins, NADH dehydrogenase ubiquinone 1 beta subcomplex subunit 8 (NADHB8), SDHB and cytochrome c oxidase 1 (MTCO1). Thus, SPARC plays a crucial role in the proliferation and differentiation of C2C12 and may be involved in the link between the ECM remodeling and mitochondrial function.</p></div

Effect of SPARC on the modulation of ECM proteins in proliferating C2C12 cells.

<p>Representative western blots for collagen 1a1 and fibronectin expressions in C2C12 myoblasts after induction/inhibition of SPARC. C2C12 myoblasts were cultured in GM. One day before adding anti-SPARC (40 μg/ml) or rSPARC (8 μg/ml), cells were trypsinezed and plated at density of 4×10⁵/well in 12-well plates. Cells were cultured in four different conditions for 48 h. Proteins extraction was performed as described above and collagen 1a1 and fibronectin protein expression levels were measured by western blot. The experiments were repeated three times (three different passages of C2C12 cells) and a representative western blot image was shown. Data were expressed as a ratio to the positive control (pooled samples). One-way ANOVA with repeated measurements was used to adjust the validations of the different passages and a contrast analysis was performed. P value was set at < 0.05 after the Bonfferoni adjustments. (a) Thirty μg of whole cell lysate proteins were loaded to measure collagen 1a1 levels in C2C12 proliferating myoblasts. Addition of SPARC induced collagen 1a1 expression, however, SPARC inhibition decreased it. (b) Five μg of the proteins from proliferating cells were loaded to measure fibronectin levels. Fibronectin expression trended to decrease after rSPARC addition and anti-SPARC increased it. Abbreviations: PBS: phosphate buffered saline, rSPARC: recombinant SPARC protein, and anti-SPARC: anti-SPARC antibody. *Significant differences between experimental conditions.</p

Modulation of mitochondrial proteins expression after exogenous induction/inhibition of SPARC in proliferating myoblasts.

<p>C2C12 cells were cultured in four different conditions which were kept in GM supplement with/without rSPARC and/or anti-SPARC for 48 h. Extracted cells lysates were prepared to perform western blot and the expression of mitochondrial OXPHOS proteins were measured using 30 μg of whole cell lysate proteins. The experiments were repeated three times (three different passages of C2C12 cells) and a representative western blot image was shown. Data were expressed as a ratio to the positive control (pooled samples). One-way ANOVA with repeated measurements was used to adjust the validations of the different passages and a contrast analysis was performed. P value was set at < 0.05 after the Bonfferoni adjustments. The addition of rSPARC (8 μg/ml) increased SDHB (a) and UQCR2 (b) proteins levels, whereas, anti-SPARC (40 μg/ml) decreased it. Abbreviations: SDHB: succinate dehydrogenase iron-sulfur subunit beta, UQCR2: ubiquinol-cytochrome c reductase core protein 2, PBS: phosphate buffered saline, rSPARC: recombinant SPARC protein, and anti-SPARC: anti-SPARC antibody. *Significant differences between experimental conditions.</p

Modulation of mitochondrial OXPHOS proteins expression after exogenous inhibition of SPARC in differentiating myobalsts.

<p>The myogenic differentiation was induced in DM supplemented with the induction/inhibition of SPARC for 5 days. DM with rSPARC (2 μg/ml) and anti-SPARC (10 μg/ml) was replaced each 2 days. Proteins extraction was performed, and extracted cells were prepared to perform western blot and the expression of mitochondrial OXPHOS proteins were measured using 30 μg of whole cell lysate proteins. The experiments were repeated three times (three different passages of C2C12 cells) and a representative western blot image was shown. Data were expressed as a ratio to the positive control (pooled samples). One-way ANOVA with repeated measurements was used to adjust the validations of the different passages and a contrast analysis was performed. P value was set at < 0.05 after the Bonfferoni adjustments. The expression of three different mitochondrial proteins was shown to be reduced after SPARC inhibition. Abbreviations: SDHB: succinate dehydrogenase iron-sulfur subunit beta, NADHB8: NADH dehydrogenase ubiquinone 1 beta subcomplex subunit 8, MTCO1: cytochrome c oxidase 1, PBS: phosphate buffered saline, rSPARC: recombinant SPARC protein, and anti-SPARC: anti-SPARC antibody. *Significant differences between experimental conditions.</p

Additional file 1: Figure S1. of Identification of the principal transcriptional regulators for low-fat and high-fat meal responsive genes in small intestine

Q_RT-PCR confirmation of SAGE and microarray. Figure S2. Comparison of the transcripts number detected in each functional classification between the serial analysis of gene expression (SAGE) and microarray. Figure S3. First network of the LF-responsive genes. Figure S4. Second network of the LF-responsive genes. Figure S5. First network of the HF-responsive genes. Figure S6. Second network of the HF-responsive genes. (DOCX 412 kb

Additional file 2: Table S1. of Identification of the principal transcriptional regulators for low-fat and high-fat meal responsive genes in small intestine

Antibody information for western blot. Table S2. LF-specific transcriptional regulators. Table S3. HF-specific transcriptional regulators. Table S4. HF-delay transcriptional regulators. Table S5. LF-HF different transcriptional regulators. Table S6. Meal-responsive transcriptional regulators. Table S7. Comparison between the serial analysis of gene expression (SAGE) and microarray in the molecular and cellular functions, networks, and transcription regulators after low-fat and high-fat meal ingestion. Table S8. Transcriptional regulators detected by SAGE. Table S9. Transcriptional regulators detected by microarray. Table S10. Normalized microarray data. (XLS 32 kb