Cell cycle and cell proliferation related genes expression variation in both skeletal muscle and heart of 11 days-old <i>Tk2</i> knockout (<i>Tk2</i>^−/−) mice.

<p><b>A</b>) Gene variation obtained by microarray analysis. SM and HE refer to each of the gene expression analyses made, in skeletal muscle and in heart. <i>n.s</i>. indicates that the gene is not significantly up- or down-regulated in the analysis. <b>B</b>) Expression of some genes was also analysed by Real-time qPCR. The <i>P</i>-values for statistical comparisons (two-tailed unpaired Student's t-test) between <i>Tk2</i>^+/+ and <i>Tk2</i>^−/− tissues are shown - *<i>P</i><0.05; **<i>P</i><0.01.</p

<i>Tk2</i>^+/+ and <i>Tk2</i>^−/− primary myoblasts culture and differentiation.

<p><b>A</b>) Primary myoblasts were isolated from 1–2 days old wild-type (<i>Tk2</i>^+/+) and <i>Tk2</i> knockout (<i>Tk2</i>^−/−) pups and cultured according to standard methods. Cultured primary myoblasts in F-10/DMEM-based primary myoblast growth medium (GM, 20% fetal bovine serum) were analysed by immunocytochemistry using anti-myogenin and anti-p21 antibodies (Abcam). Nuclei have been stained with DAPI (Sigma). <b>B</b>) Isolated primary myoblasts capacity to differentiate in myotubes was tested by changing them to differentiation medium (DM - DMEM with 5% horse serum). Immunocytochemistry was performed using anti-myogenin and anti-p21 antibodies (Abcam). Nuclei have been stained with DAPI (Sigma). Pictures were taken 2 days after myoblasts have been transferred to DM.</p

Gene expression variation of some genes involved in cell cycle arrest in both skeletal muscle and heart of 11 days-old <i>Tk2</i> knockout (<i>Tk2</i>^−/−) mice.

<p><b>A</b>) Expression values obtained in the microarray analysis for some genes involved in cell cycle arrest at G1 or G2. SM and HE refer to each of the gene expression analyses made, in skeletal muscle and in heart. <i>n.s.</i> indicates that the gene is not significantly up- or down-regulated in the analysis. <b>B</b>) Expression of <i>Cdkn1a</i> (p21) was also analysed by Real-time qPCR for both tissues. The <i>P</i>-values for statistical comparisons (two-tailed unpaired Student's t-test) between <i>Tk2</i>^+/+ and <i>Tk2</i>^−/− tissues are shown - *<i>P</i><0.05; **<i>P</i><0.01.</p

<i>Tk2</i>^+/+ and <i>Tk2</i>^−/− mice skeletal muscle and heart mitochondria ultrastructure.

<p>Transmission electron microscopy images of skeletal muscle (<b>A</b>) and heart (<b>B</b>) sections isolated from wild-type (<i>Tk2</i>^+/+) and <i>Tk2</i> knockout (<i>Tk2</i>^−/−) 14 days-old mice. Mitochondria are indicated with solid arrows in the pictures.</p

mtDNA copy number per diploid nucleus in skeletal muscle and heart of <i>Tk2</i>^+/+ and <i>Tk2</i>^−/− pups at 4, 8 and 14 days after birth.

<p>For each time point, data from at least 3 different mice have been used (<i>n</i>≥3). The <i>P</i>-values for statistical comparisons (two-tailed unpaired Student's t-test) between <i>Tk2</i>^+/+ and <i>Tk2</i>^−/− tissues are shown - *<i>P</i><0.05;</p>**<p><i>P</i><0.01.</p

Gene expression deregulation in 11 days-old <i>Tk2</i> knockout (<i>Tk2</i>^−/−) skeletal muscle and heart comparing to wild-type (<i>Tk2</i>^+/+) tissues from mice with the same age.

<p><b>A</b>) Considering only genes with at least 2.0-fold variation in expression and with a statistically significant <i>P</i>-value (<i>P</i>-value<0.05), the number of up- and down-regulated genes in <i>Tk2</i>^−/− skeletal muscle and heart, comparing to the same tissues in <i>Tk2</i>^+/+ mice, are indicated. The numbers of similar genes that were up- or down-regulated in both analyses are indicated in the intersection of the Venn diagrams. <b>B</b>) In the group of differentially expressed genes in skeletal muscle and heart, statistically significant enrichment of genes belonging to specific KEGG pathways was detected using the Pathway analysis from the Expander software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053698#s4" target="_blank">methods</a>). Pathways significantly enriched in the group of up-regulated genes are marked with an upwards arrow (↑) and those enriched in the group of down-regulated genes are marked with a downwards arrow (↓). SM and HE indicate each of the gene expression analyses done, in skeletal muscle and in heart, respectively.</p

Body and organs weights in 14 days-old <i>Tk2</i>^+/+ and <i>Tk2</i>^−/− mice.

<p>For each measurement, data from at least 3 different mice have been used (<i>n</i>≥3).The <i>P</i>-values for statistical comparisons (two-tailed unpaired Student's t-test) between <i>Tk2</i>^+/+ and <i>Tk2</i>^−/− body or tissues weights are shown - **<i>P</i><0.01;</p>***<p><i>P</i><0.001.</p

dNTP pool maintenance related genes expression variation in both skeletal muscle and heart of 11 days-old <i>Tk2</i> knockout (<i>Tk2</i>^−/−) mice.

<p><b>A</b>) Gene variation obtained by microarray analysis. SM and HE refer to each of the gene expression analyses made, in skeletal muscle and in heart. <i>n.s.</i> indicates that the gene is not significantly up- or down-regulated in the analysis. <b>B</b>) Expression of deoxyribonucleoside kinases genes was also analysed by Real-time qPCR. The <i>P</i>-values for statistical comparisons (two-tailed unpaired Student's t-test) between <i>Tk2</i>^+/+ and <i>Tk2</i>^−/− tissues are shown - *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

Percentage of <i>Tk2^+/+</i> and <i>Tk2^−/−</i> positive cells for myogenin and p21 and of nuclei contained within multinucleated myotubes (fusion index) in growth and differentiation conditions.

<p>The frequency of each marker was calculated from double immunofluorescence experiments of <i>Tk2^+/+</i> and <i>Tk2^−/−</i> cells maintained in growth medium or exposed to differentiation medium for 1 or 2 days. The percentage of myogenin or p21 positives and the fusion index were expressed relative to the total number of DAPI stained nuclei in 10 different microscope fields (about 700–800 nuclei counted for each sample).</p

Additional file 2: of Inhibition of glutamate oxaloacetate transaminase 1 in cancer cell lines results in altered metabolism with increased dependency of glucose

Figure S2. Rescue of GOT1 down-regulated and GOT1-null cells by oxaloacetate. Relative cell viabilities after 8 h (a) and 24 h (b) in wild type 143B cells and 8 h (c) and 24 h (d) in wild type A549 cells. Relative cell viabilities after 8 h (e) and 24 h (f) in GOT1 siRNA knock-down A549 cells. Rescue of GOT1-null 143B cells with OAA at different concentrations upon glucose deprivation (g). Mean ± s.d. from 3 independent experiments. One-way ANOVA test was performed. *** p < 0.001; ** p < 0.01;* p < 0.05. NS: not significant. (TIF 230 kb