Protein interaction networks under the overexpression condition.

<p>(A) Biggest network under the overexpression condition, containing 108 genes. (B) Secondary interaction networks of MYH10, containing 47 genes. (C) Network of genes that directly interact with MYH10, containing 11 genes.</p

Enriched biological process (BP) terms.

<p>(A) BP terms enriched under the knockdown condition (NC vs Inter). (B) BP terms enriched under the overexpression condition (Control vs Over).</p

Myoz3 overexpression results in fast muscle specific gene up-regulated but not slow muscle specific gene.

<p>(A) Slow muscle specific gene <i>MYH7B</i>. (B) Fast muscle specific gene <i>MYH1F</i>. NC; non-specific control siRNA. Inter; <i>Myoz3</i> siRNA. Control; empty vector. Over; <i>Myoz3</i> overexpression vector. The relative expression level of data was presented by bar plots with error bars represent SEM. All experiments were replicated three times. Error bars represent SEM. *P<0.05, **P<0.01, ***P<0.001.</p

Validation of differentially expressed genes by qRT-PCR.

<p>The relative expression level of data was presented by bar plots with error bars represent SEM. All experiments were replicated three times. (A) <i>MYL9</i>, (B) <i>MYL4</i> (C) <i>PKM</i>, (D) <i>MPRIP</i>, (E) PDLIM1, (F) <i>MYH10</i>, (G) <i>MYLK2</i>, (H) <i>PPM1J</i>, (I) <i>ECM2</i>, (J) <i>OASL</i>, (K) <i>FAP</i>, (L) <i>CaN</i>, (M) <i>ITGA8</i>. *P<0.05, **P<0.01, ***P<0.001, N.S. means not significant.</p

Experimental design and overall results.

<p>The expression levels of the top 30 differentially expressed genes are shown in log CPM (copies per million). NC (non-specific control).</p

<i>Myoz3</i> siRNA and overexpression vector screening.

<p>(A) <i>Myoz3</i> expression level in breast muscle, CEFs and Myoblasts. (B) <i>Myoz3</i> relative expression level in CEFs after transfection with non-specific siRNA (Control), and candidate siRNA. (C) Myoz3 relative expression level in myoblasts after transfection with non-specific siRNA (Control), and candidate siRNA. (D) <i>Myoz3</i> relative expression level in CEFs after transfection with empty vector and overexpression vector (Over). The error bars represent SEM; all experiments were replicated three times. *P<0.05, **P<0.01, ***P<0.001.</p

Proliferation by CCK-8 under both <i>Myoz3</i> knockdown and overexpression conditions.

<p>(A) 24 hours after CEFs transfected with empty vector (Control) or overexpression vector, non-specific siRNA (NC) or <i>Myoz3</i> siRNA (Inter). (B) 48 hours after CEFs transfected with empty vector (Control) or overexpression vector, non-specific siRNA (NC) or <i>Myoz3</i> siRNA (Inter). (C) 24 hours after Myoblast transfected with empty vector (Control) or overexpression vector, non-specific siRNA (NC) or <i>Myoz3</i> siRNA (Inter). (D) 48 hours after myoblasts transfected with empty vector (Control) or overexpression vector, non-specific siRNA (NC) or <i>Myoz3</i> siRNA (Inter).</p

Primers for qRT-PCR.

<p>Primers for qRT-PCR.</p

The 3^rd sample of Inter is abnormal due to inefficient knockdown.

<p><b>(A)</b> An MDS plot of NC and Inter illustrates the abnormality of the 3^rd Inter sample. <b>(B)</b> The expression level was not sufficiently knocked down in the 3^rd Inter group. The genes that were differentially expressed between the two experiment conditions are presented. The error bars indicate SEM for three replicates (qRT-PCR). Significance was not calculated because to data for each bar do not represent biological replicates.</p

Transcriptomic analysis of chicken <i>Myozenin 3</i> regulation reveals its potential role in cell proliferation

<div><p>Embryonic muscle development and fibre type differentiation has always been a topic of great importance due to its impact on both human health and farm animal financial values. <i>Myozenin3</i> (<i>Myoz3</i>) is an important candidate gene that may regulate these processes. In the current study, we knocked down and overexpressed <i>Myoz3</i> in chicken embryonic fibroblasts (CEFs) and chicken myoblasts, then utilized RNA-seq technology to screen genes, pathways and biological processes associated with <i>Myoz3</i>. Multiple differentially expressed genes were identified, including <i>MYH10</i>, <i>MYLK2</i>, <i>NFAM1</i>, <i>MYL4</i>, <i>MYL9</i>, <i>PDZLIM1</i>; those can in turn regulate each other and influence the development of muscle fibres. Gene ontology (GO) terms including some involved in positive regulation of cell proliferation were enriched. We further validated our results by testing the activity of cells by cell counting kit-8(CCK-8) and confirmed that under the condition of <i>Myoz3</i> overexpression, the proliferation rate of CEFs and myoblasts was significantly upregulated, in addition, expression level of fast muscle specific gene was also significantly upregulated in myoblasts. Pathway enrichment analysis revealed that the PPAR (Peroxisome Proliferator-Activated Receptor) pathway was enriched, suggesting the possibility that <i>Myoz3</i> regulates muscle fibre development and differentiation through the PPAR pathway. Our results provide valuable evidence regarding the regulatory functions of <i>Myoz3</i> in embryonic cells by screening multiple candidate genes, biological processes and pathways associated with <i>Myoz3</i>.</p></div