Experimental set up and analyses of expression profiling data.

<p>(A) Schematic overview of sampling. MB: myoblasts cultured in growth medium. MT: myotubes cultured in differentiation medium. TNF: myotubes cultured in differentiation medium with TNF-α. IGF: myotubes cultured in differentiation medium with IGF-1. (B) Gene expression data were analyzed by hierarchical clustering, principal component analysis (PCA), and dynamic PCA among others. Moreover, the profiling data were clustered by applying the self-organized tree algorithm (SOTA) as well as pathway association enrichment analyses. Furthermore, genes adversely regulated by differentiation and TNF-α were identified. Finally, TNF-α-regulated genes were compared with results of other studies. The study identified novel insights into the gene expression mechanisms and kinetics of early skeletal myocyte differentiation and how this is modified by TNF-α.</p

Genes describing the treatment effects.

<p>Nonambigous genes identified by dynamic principal component analysis (group selection myoblasts) of gene expression profiling data after 4 h, 12 h, or 24 h of differentiation or TNF-α or IGF1 treatment identified genes that were sufficient for separation of treatment groups by principal components as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139520#pone.0139520.g002" target="_blank">Fig 2C, 2F and 2I</a>.</p><p>Genes describing the treatment effects.</p

Transcriptomic signatures of myoblast differentiation and TNF-α or IGF1 treatment.

<p>Principal component analyses (PCA) of mRNA expression profiling data after (A) 4 h, (D) 12 h, or (G) 24 h of induction of differentiation with TNF-α, IGF1, or control treatment showing nonambiguous genes are depicted. Axes show principal components (PC) 1, PC 2, and PC 3. PCA revealed separation of treatment groups. Light blue indicates myoblasts, green marks myotubes, red distinguishes myotubes treated with TNF-α, whereas dark blue indicates myotubes treated with IGF1. The proportions of variance for the first six components of principal component analysis are depicted for the effect of (B) 4 h, (E) 12 h, and (H) 24 h after induction of differentiation and the respective treatments. Most of the variance is described by PC 1 followed by PC 2 and PC 3. PC 1 explaines most of the variance of myocyte differentiation while PC 2 represented most of the variance induced by TNF-α whereas PC 3 characterized most of the variance caused by IGF1 treatment. Moreover, results from dynamic principal component analyses (dPCA) (group selection myoblasts) are shown for gene expressions (C) 4 h, (F) 12 h, and (I) 24 h after induction of differentiation and treatment. DPCA identified a minimal subset of genes, which could describe the treatment effects (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139520#pone.0139520.t002" target="_blank">Table 2</a>) and separate the effects by principal components.</p

Western Blot analysis of differentially expressed genes.

<p>(A) Chk1, (B) Emi1/Fbxo5, (C) Mybl2 protein were detected by western blot analysis. Histone H3 served as the normalization control. Murine mouse muscle cells were cultured for 48 h in growth medium (lane 1), differentiation medium (lane 2), or differentiation medium supplemented with TNF-α (lane 3) or IGF1 (lane 4), respectively. (B) The specificity of the double band between 40 and 50 kDa was confirmed by peptide competition of the Emi/Fbxo5 antibody epitope (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139520#pone.0139520.s004" target="_blank">S4 Fig</a>).</p

Novel genes and pathways in skeletal myocyte differentiation and TNF-α response.

<p>We identified genes and pathway associations, which have not been described before in skeletal myocyte differentiation or have been reported to have a different regulation than the one observed in the current study. A plus indicates upregulation during differentiation and a minus indicates downregulation. Moreover, we show genes which are inversely regulated by TNF-α, but have not been defined before, to be regulated in skeletal myocyte differentiation and response to TNF-α.</p

Pathway enrichment analysis of differentially expressed genes.

<p>Signal transduction pathway associations, which were enriched after 4 h (“induction of differentiation”/immediate response), and 12 h (very early differentiation) of treatment, and 24 h (early differentiation) treatment, are depicted. The effects of differentiation without or with TNF-α or with IGF1 compared with TNF-α treatment are shown. Pathway enrichment was based on cocitation with a p value cutoff of <0.01. Genes within significantly enriched pathways are listed. In addition, it is indicated in which SOTA cluster a pathway is enriched. Pathways highlighted in bold are retrieved in enrichment analyses of genes identified by principal component analysis which are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139520#pone.0139520.t001" target="_blank">Table 1</a>.</p><p>Pathway enrichment analysis of differentially expressed genes.</p

Pathway enrichment analysis of genes separated by principal components.

<p>Signal transduction pathway associations of genes which are separated by principal component analysis after 4-h, 12-h, or 24-h treatment are depicted. Principal component one separates the effect of differentiation, whereas principal component two represents the effect of TNF-α treatment. Finally, principal component three separates the effect of IGF1 treatment versus control myotubes. Pathway enrichment was based on cocitation with a p value cutoff of <0.01. Genes within significantly enriched pathways are listed. Pathways highlighted in bold are retrieved in enrichment analyses of differentially expressed genes which are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139520#pone.0139520.t003" target="_blank">Table 3</a>.</p><p>Pathway enrichment analysis of genes separated by principal components.</p

TNF-α inversely regulated differentiation genes.

<p>List of genes upregulated by differentiation but downregulated because of TNF-α treatment or vice versa. Log2 ratios indicate the order of magnitude of differential expression. Enriched signal transduction pathway associations in which the respective gene is involved during differentiation or TNF-α treatment are shown. The literature background indicates whether the gene has been published in skeletal muscle differentiation (SkMDiff), skeletal muscle (SkM), heart muscle, smooth muscle, or muscle progenitor cells (M), or whether it has not been described in muscle (new).</p><p>TNF-α inversely regulated differentiation genes.</p

Coregulation of gene sets during myogenic differentiation as well as TNF-α and IGF1 treatment.

<p>Self-organizing tree algorithm (SOTA) analysis of gene expression data within the first 72 h of differentiation as well as TNF-α and IGF1 treatment revealed the following clusters of gene sets: (A) cluster A contained 335 genes upregulated during very early differentiation and (B) cluster B comprised 351 genes upregulated during later differentiation. (C) Genes totaling 172, which were downregulated during very early differentiation, were summarized by cluster C. (D) Genes induced by TNF-α but suppressed during late myotubes were visualized by cluster C implying eight genes. (E) Cluster E included 40 genes specifically induced by TNF-α. (F) Forty genes downregulated later during differentiation were represented by cluster F. Gene identities and signal transduction pathway associations of genes within the individual SOTA clusters are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139520#pone.0139520.s008" target="_blank">S4 Table</a>. Furthermore, clusters G, H, and I bear the minority of genes and are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139520#pone.0139520.s002" target="_blank">S2 Fig</a>.</p

LDH-B does not show BIAcore binding signal with GNE.

<p>[A] Sensorgram of the interaction of GNE with 5 µM LDH-B, compared to 5 µM α-actinin 1. [B] Sensorgram showing the effect of 2 µM LDH-B on the interaction of GNE with 0.5 µM α-actinin 1 [Resp. Diff., response difference; RU, response units].</p