Additional file 1: of Assessing the activity of nonsense-mediated mRNA decay in lung cancer

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Additional file 4: of Assessing the activity of nonsense-mediated mRNA decay in lung cancer

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Additional file 2: of Assessing the activity of nonsense-mediated mRNA decay in lung cancer

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DNA Methylation Variation Trends during the Embryonic Development of Chicken

<div><p>The embryogenesis period is critical for epigenetic reprogramming and is thus of great significance in the research field of poultry epigenetics for elucidation of the trends in DNA methylation variations during the embryonic development of birds, particularly due to differences in embryogenesis between birds and mammals. Here, we first examined the variations in genomic DNA methylation during chicken embryogenesis through high-performance liquid chromatography using broilers as the model organism. We then identified the degree of DNA methylation of the promoters and gene bodies involved in two specific genes (<i>IGF2</i> and <i>TNF-α</i>) using the bisulfite sequencing polymerase chain reaction method. In addition, we measured the expression levels of <i>IGF2</i>, <i>TNF-α</i> and DNA methyltransferase (<i>DNMT</i>) 1, 3a and 3b. Our results showed that the genomic DNA methylation levels in the liver, heart and muscle increased during embryonic development and that the methylation level of the liver was significantly higher in mid-anaphase. In both the muscle and liver, the promoter methylation levels of <i>TNF-α</i> first increased and then decreased, whereas the gene body methylation levels remained lower at embryonic ages E8, 11 and 14 before increasing notably at E17. The promoter methylation level of <i>IGF2</i> decreased persistently, whereas the methylation levels in the gene body showed a continuous increase. No differences in the expression of <i>TNF-α</i> were found among E8, 11 and 14, whereas a significant increase was observed at E17. <i>IGF2</i> showed increasing expression level during the examined embryonic stages. In addition, the mRNA and protein levels of <i>DNMTs</i> increased with increasing embryonic ages. These results suggest that chicken shows increasing genomic DNA methylation patterns during the embryonic period. Furthermore, the genomic DNA methylation levels in tissues are closely related to the genes expression levels, and gene expression may be simultaneously regulated by promoter hypomethylation and gene body hypermethylation.</p></div

Primer sequences for bisulfite sequencing polymerase chain reaction analysis.

<p>Primer sequences for bisulfite sequencing polymerase chain reaction analysis.</p

DNA methylation patterns of the tumer necrosis factor-alpha (<i>TNF-α</i>) and insulin-like growth factor 2 (<i>IGF2</i>) gene bodies in the livers of broilers.

<p>The analytic method was the bisulfite sequencing polymerase chain reaction. Each line represents an individual bacterial clone, and each circle represents a single CpG dinucleotide. Open circles indicate unmethylated CpGs, and black circles indicate methylated CpGs. (a) <i>TNF-α</i> detected in the liver. (b) <i>IGF2</i> detected in the liver. (c) The methylation levels of the <i>TNF-α</i> and <i>IGF2</i> gene bodies in the histogram.</p

Variations in the genomic DNA methylation levels in broilers during the embryogenesis.

<p>The samples examined from embryonic ages (E) 2 to 7 were a mixture of the whole embryo; from E 9 to 20, the examined samples were separated into the heart, liver and muscle tissues. 5-mdC: 5-methyldeoxycytosine.</p

Schematic representation of the proximal promoter region and part of the first intron of tumer necrosis factor-alpha (<i>TNF-α</i>) and insulin-like growth factor 2 (<i>IGF2</i>) for predicting regions with a high GC content.

<p>The proximal promoter regions of <i>TNF-α</i> (a, -2000 to +1 base pairs [bp]) and <i>IGF2</i> (c, -2000 to +1 bp) and part of the first introns of <i>TNF-α</i> (b, +500 to +2500 bp) and <i>IGF2</i> (d, +5000 to +7000 bp) (modified output of MethPrimer program [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159230#pone.0159230.ref020" target="_blank">20</a>]) were used to predict regions of high GC content. A dashed black line indicates the GC percentage as represented on the y-axis, and the x-axis denotes the bp position on the <i>TNF-α</i> and <i>IGF2</i> 5'-untranslated and gene body regions. The arrows indicate the transcriptional start sites (TSS). The coordinates are given in relation to the translation initiation site (shown as +1); vertical red lines indicate the relative positions of CpG dinucleotides; solid lines depict the locations of the PCR primers. Input sequences are shown as the bold region of the intermediate solid blue line; the untranslated and translated exons of <i>TNF-α</i> and <i>IGF2</i> are indicated by light blue and dark blue bars, respectively. The start sites, end sites and numbers of CG loci of the PCR products are all shown under each bisulfite PCR primer.</p

Protein expression levels of insulin-like growth factor 2 (IGF2), tumor necrosis factor-alpha (TNF-α) and DNA methyltransferases (DNMT) in the muscles and livers of broilers.

<p>(a) Protein expression levels of the target proteins in the livers. (b) Protein expression levels of the target proteins in the muscles.</p

Relative mRNA levels of DNA methyltransferases (<i>DNMT</i>) 1, <i>DNMT3a</i> and <i>DNMT 3b</i> in the muscles and livers of broilers.

<p>(a): <i>DNMT1</i>, (b): <i>DNMT3a</i>, (c): <i>DNMT 3b</i>. The data represent the means with standard errors (n = 6). Bars with different letters differed significantly (<i>P</i> < 0.05).</p