Table 1.
Body and organs weights in 14 days-old Tk2+/+ and Tk2−/− mice.
Table 2.
mtDNA copy number per diploid nucleus in skeletal muscle and heart of Tk2+/+ and Tk2−/− pups at 4, 8 and 14 days after birth.
Figure 1.
Tk2+/+ and Tk2−/− mice skeletal muscle and heart mitochondria ultrastructure.
Transmission electron microscopy images of skeletal muscle (A) and heart (B) sections isolated from wild-type (Tk2+/+) and Tk2 knockout (Tk2−/−) 14 days-old mice. Mitochondria are indicated with solid arrows in the pictures.
Figure 2.
Gene expression deregulation in 11 days-old Tk2 knockout (Tk2−/−) skeletal muscle and heart comparing to wild-type (Tk2+/+) tissues from mice with the same age.
A) Considering only genes with at least 2.0-fold variation in expression and with a statistically significant P-value (P-value<0.05), the number of up- and down-regulated genes in Tk2−/− skeletal muscle and heart, comparing to the same tissues in Tk2+/+ 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) 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 methods). 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.
Figure 3.
Gene expression variation of some genes involved in cell cycle arrest in both skeletal muscle and heart of 11 days-old Tk2 knockout (Tk2−/−) mice.
A) 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. n.s. indicates that the gene is not significantly up- or down-regulated in the analysis. B) Expression of Cdkn1a (p21) was also analysed by Real-time qPCR for both tissues. The P-values for statistical comparisons (two-tailed unpaired Student's t-test) between Tk2+/+ and Tk2−/− tissues are shown - *P<0.05; **P<0.01.
Figure 4.
Cell cycle and cell proliferation related genes expression variation in both skeletal muscle and heart of 11 days-old Tk2 knockout (Tk2−/−) mice.
A) Gene variation obtained by microarray analysis. SM and HE refer to each of the gene expression analyses made, in skeletal muscle and in heart. n.s. indicates that the gene is not significantly up- or down-regulated in the analysis. B) Expression of some genes was also analysed by Real-time qPCR. The P-values for statistical comparisons (two-tailed unpaired Student's t-test) between Tk2+/+ and Tk2−/− tissues are shown - *P<0.05; **P<0.01.
Figure 5.
dNTP pool maintenance related genes expression variation in both skeletal muscle and heart of 11 days-old Tk2 knockout (Tk2−/−) mice.
A) Gene variation obtained by microarray analysis. SM and HE refer to each of the gene expression analyses made, in skeletal muscle and in heart. n.s. indicates that the gene is not significantly up- or down-regulated in the analysis. B) Expression of deoxyribonucleoside kinases genes was also analysed by Real-time qPCR. The P-values for statistical comparisons (two-tailed unpaired Student's t-test) between Tk2+/+ and Tk2−/− tissues are shown - *P<0.05; **P<0.01; ***P<0.001.
Figure 6.
Tk2+/+ and Tk2−/− primary myoblasts culture and differentiation.
A) Primary myoblasts were isolated from 1–2 days old wild-type (Tk2+/+) and Tk2 knockout (Tk2−/−) 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) 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.
Table 3.
Percentage of Tk2+/+ and Tk2−/− positive cells for myogenin and p21 and of nuclei contained within multinucleated myotubes (fusion index) in growth and differentiation conditions.
Figure 7.
Tk2+/+ and Tk2−/− primary myoblasts characterization.
A) Growth of Tk2+/+ and Tk2−/− myoblasts in F-10/DMEM-based primary myoblast growth medium was determined by XTT proliferation assay (see methods). Data points represent mean ± SD of measurements obtained from 4 independent myoblast cultures (n = 4). The P-values for statistical comparisons (two-tailed unpaired Student's t-test) between Tk2+/+ and Tk2−/− data points are shown - **P<0.01; ***P<0.001. B) Thymidine kinase activity measured in both Tk2+/+ and Tk2−/− cultured myoblasts (n = 2). C) mtDNA copy number of Tk2+/+ and Tk2−/− cultured myoblasts was measured by Real-time PCR in growth (GM) and differentiation conditions (DM, 8 days). D) Flow cytometry analysis of Tk2+/+ and Tk2−/− myoblasts cell cycle, 24 hours after cells have been plated. Data were obtained from 3 independent experiments (n = 3) and were analysed using the ModFit LT software, in order to obtain the percentages of cells in G1, S and G2/M cell cycle phases. E) Expression of several genes in Tk2+/+ and Tk2−/− growing myoblasts (GM) and differentiated myotubes (DM, 6 days) analysed by Real-time qPCR. The P-values for statistical comparisons (two-tailed unpaired Student's t-test) between Tk2+/+ and Tk2−/− cells are shown - *P<0.05; **P<0.01; ***P<0.001. F) Western-blot analysis of the expression of p21, E2F1, Rb1 and VDAC proteins in Tk2+/+ and Tk2−/− differentiated myotubes (DM, 6days).