Primer sequences for real-time and semi-quantitative RT-PCR.

<p>Primer sequences for real-time and semi-quantitative RT-PCR.</p

Comparison of the h<i>FTSJ2</i> mRNA expression levels in two lung cancer sublines (CL1-0 and CL1-5).

<p>(<b>A</b>) Morphology of CL1-0 and CL1-5 cells. (<b>B</b>) Determination of the h<i>FTSJ2</i> mRNA expression levels in the CL1-0 and CL1-5 cells in triplicate. (<b>C</b>) Relative quantification of the h<i>FTSJ2</i> mRNA expression. <i>GAPDH</i> mRNA was used as an internal control. The values are equal to = the means±SE; n = 3; **<i>P</i><0.01 vs. the non-heat shock group.</p

Protein sequence alignment of <i>E. coli</i> RrmJ with its FTSJ2 orthologs in 7 different species.

<p>The α-helices and β-strands were based on the RrmJ protein structure (PDB code: 1EIZ). The stars and triangles indicate the K-D-K-E catalytic center and the SAM binding residues in RrmJ, respectively. The residues with identical and similar chemical properties are highlighted in black and gray, respectively.</p

Subcellular localization of human FTSJ2 in TE671 and HepG2 cells.

<p>(<b>A</b>) Schematic of the hFTSJ2 over-expression vector. (<b>B</b>) Over-expression of the hFTSJ2 protein in the TE671-h<i>FTSJ2</i> and HepG2-h<i>FTSJ2</i> stable clones. (<b>C</b>) Immunofluorescence staining with anti-hFTSJ2 (green), MitoTracker for mitochondria (red), and DAPI for nuclei (blue) in the TE671-h<i>FTSJ2</i> and HepG2-h<i>FTSJ2</i> cells. (<b>D</b>) Mitochondrial localization of the hFTSJ2 protein in non-transfected TE671 cells. VDAC and MEK-1 were used as the mitochondrial and cytosolic fraction controls, respectively, in the Western blot analysis.</p

Body weight, survival time, and aberrant methylation status of imprinted genes in wild-type and of cloned pigs.

1<p>The body weight of control newborn piglets is an average value from 10 litters (98 piglets) of same-age piglets of the nuclear donor pig breed. WT: wild-type. M: maternally imprinted genes (<i>H19</i> and <i>IGF2R</i>). P: paternally imprinted genes (<i>IGF2</i> and <i>INS</i>).</p

Methylation status of the <i>H19</i> putative DMR in cloned and wild-type pigs.

<p>(A) Schematic of the putative DMR of <i>H19</i>, located in the promoter. The Southern blot hybridization probe is shown as a black box. P1, P2 and P3 indicate three CTCF-binding sites of the putative DMR. (B) Southern blot hybridization results in the <i>H19</i> DMR in cloned pigs. The level-1 methylation percentage was calculated by the bands of 2643 bp, 967 bp, and 689 bp. The level-2 methylation percentage was calculated by the bands of 967 bp and 689 bp. (C) Methylation-specific PCR analysis of the <i>H19</i> promoter region in cloned pigs. The black arrow shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032812#pone-0032812-g002" target="_blank">Figure 2A</a> indicates the primer sets used in the MS-PCR assay. The number below the panel indicates the methylation percentage. Br: brain; Ea: ear; He: heart; Ki: kidney; Li: liver; Lu: lung; Mu: muscle; Pl: placenta; Um: umbilical cord; B: blood; S: blood treated with <i>Sss</i>I; W: ddH₂O.</p

Dissection of the methylation status of the putative <i>INS</i> DMR in cloned and wild-type pigs.

<p>(A) A schematic diagram of the pig <i>INS</i> gene showing the relative positions of promoter, exon 1 and CpG islands. The black arrow indicates the location of the primer set. The striped box indicates the putative DMR. The horizontal line indicates the CpG site. The PCR product was digested with <i>Bst</i>UI. (B) The putative DMR of the maternally imprinted <i>INS</i> gene in cloned pigs and wild-type pigs was analyzed by COBRA. The numbers under the figure in the WT panel indicate the average methylation percentage of three wild-type pigs. (C) Bisulfite sequencing of the <i>INS</i> putative DMR in WT ear, CP4 liver, and CP3 ear. Open and closed circles indicate unmethylated and methylated CpG sites, respectively. The top number indicates the CpG site position of the analyzed <i>INS</i> putative DMR. The bottom line indicates the <i>Bst</i>UI recognition site. Fourteen clones of each tissue were sequenced. The methylation percentage calculations were divided into three parts: CpG sites 1 to 20 (all), CpG sites 15 to 20, and CpG sites 13 to 20. For example, WT ear showed a total methylation percentage of 75.7 (212 methylated CpG sites/20 CpG sites ×14 clones = 75.7%). M1: Bio 100 DNA ladder; M2: 500 bp DNA ladder; Br: brain; Ea: ear; He: heart; Ki: kidney; Li: liver; Lu: lung; Mu: muscle; Pl: placenta; Um: umbilical cord; B: blood; S: blood treated with <i>Sss</i>I; W: ddH₂O.</p

Methylation status of the <i>IGF2R</i> putative DMR of intron 2 in cloned pigs and wild-type pigs by COBRA analysis.

<p>(A) Schematic diagram showing the distribution of CpG sites of the pig <i>IGF2R</i> gene. (B) COBRA data showing the methylation status of the <i>IGF2R</i> intron 2 putative DMR in control and wild-type pigs. The numbers under the figure in the control panel indicate the average methylation percentage of three wild-type pigs. Br: brain; Ea: ear; He: heart; Ki: kidney; Li: liver; Lu: lung; Mu: muscle; Pl: placenta; Um: umbilical cord; B: blood; S: blood treated with <i>Sss</i>I; W: ddH₂O.</p

The aberrant methylation of the <i>H19</i>, <i>INS</i> and <i>IGF2</i> genes and their mRNA expression levels in cloned pigs.

<p>(A) DNA methylation statuses of <i>H19</i>, <i>INS</i>, and <i>IGF2</i> in CP2 ear, CP4 ear, and CP3 ear, respectively. (B) mRNA expression levels of <i>H19</i>, <i>INS</i> and <i>IGF2</i> gene in CP2 ear, CP4 ear, and CP3 ear, respectively. (C) mRNA levels from panel B relative to <i>β-actin</i>.</p

Identification of the putative DMRs of four imprinted genes and their normal differential methylation patterns in the different tissues of wild-type pigs.

<p>(A) Schematic of CpG site distributions in the putative DMRs of four imprinted genes, <i>IGF2</i>, <i>H19</i>, <i>INS</i>, and <i>IGF2R</i>. Vertical black lines represent each CpG site. Horizontal gray bars represent analyzed regions. Horizontal reticular bars represent putative DMRs in the imprinted genes. Horizontal solid black bars represent probes used for Southern blot hybridization. The upper line indicates the scale bar for DNA length. The putative DMR of <i>H19</i> is located between nt 30,856 and nt 33,489 (GenBank accession no. AY044827). The CpG island of <i>H19</i> corresponds to our designed probe, which ranges from nt 31,411 to nt 31,818. The putative DMR1 of pig <i>IGF2</i> is located between exon 3 and exon 4 and ranges from nt 17,620 to nt 18,796 (GenBank accession no. AY044828). The CpG island of <i>IGF2</i> corresponds to our designed probe, which ranges from nt 17,733 to nt 18,048. The putative DMR2 of <i>IGF2</i> is located in exon 9, nt 27,441 to nt 27,819 (GenBank accession no. AY242102.1). The putative DMR of pig <i>INS</i> is located between nt 1,456 and nt 2,323, and the probe ranges from nt 1,663 to nt 1,986 (GenBank accession no. AY242112). The putative DMR of <i>IGF2R</i> is located between exon 2 and exon 3 (GenBank accession no. AF339885). (B) The normal differential methylation patterns of the four imprinted genes in several tissues of wild-type pigs. The methylation statuses of <i>IGF2</i>, <i>INS</i>, and <i>IGF2R</i> were assayed by COBRA. The methylation status of <i>H19</i> was assayed with Southern blot analysis. The numbers under the images indicate the average methylation percentage in the different tissues of three wild-type pigs (n = 3). Mu: muscle; He: heart; Ea: ear; Li: liver; Lu: lung; Ki: kidney; Br: brain; Pl: placenta.</p