Analysis of body weight, contents of DNA, RNA and protein in <i>M. quadriceps femoris</i> of Fzt:DU (29 weeks) and DU6P (11 and 29 weeks) female mice (n = 5).

<p>a,b - different superscripts indicate significant differences (p<0.05);</p>*<p>- significantly different if compared to 11-week DU6P or Fzt:DU, respectively as evaluated using the Wilcoxon-signed rank test.</p><p>Furthermore the non-polysomal and polysomal RNA fraction in <i>M. quadriceps femoris</i> of Fzt:DU (29 weeks) and DU6P (11 and 29 weeks) female mice (n = 4) was analysed.</p

A: Serum IGF-I levels in female Fzt:DU and DU6P mice over life time (n = 8 per age group).

<p>B: Expression of IGF-II precursor (23 kDa) in muscle tissues from 11-, 29- and 54-week female Fzt:DU and DU6P mice (n = 10 per age group). The results are normalized for the signal intensities as detected by Coomassie blue staining of the membranes used for Western immuno detection. Data are expressed as percent of female 11-week Fzt:DU mice (100%). The error bars represent SEM.</p

Long-term phenotype selection for high protein mass in mice.

<p>A: Phenotypes of female Fzt:DU and DU6P mice at the age of 11, 29 and 42 weeks. B: Success for the selected trait (protein mass) in the course of long-term selection in DU6P mice versus unselected control mice (Fzt:DU). C: Dissected <i>M. quadriceps femoris</i> of 11, 29 and 42-week female Fzt:DU and DU6P mice. D: Longitudinal body weights in female DU6P mice versus Fzt:DU mice. E and F: Relative weights of <i>M. quadriceps femoris</i> and carcasses from female Fzt:DU and DU6P mice (n = 15; p<0.03 for all comparisons of age-matched mouse lines). The sample number (n) depicts the number of samples per age group, the error bars represent SEM.</p

In mid-aged female DU6P mice inhibition of PTEN by means of reduced PTEN expression and increased PTEN phosphorylation correlates with higher levels of AKT phosphorylation at Ser-473, inactivation of GSK3ß on one hand and higher levels of S6K on the other.

<p>An increase of muscle mass is due to higher levels of muscle glycogen and an increased rate of protein synthesis in female 29-week DU6P mice.</p

Analysis of signal transduction in muscle lysates from 11-, 29- and 54-week female DU6P and Fzt:DU mice.

<p>The Western blot inserts show phosphorylated (Ser-473) and total expression of AKT from one representative experiment, whereby all samples were studied on the identical membrane. Each Western blots were performed three times. Thus a total of 9 different animals was included in the bar chart for each timepoint. Specific activation was calculated from the ratios of phosphorylated versus total AKT (n = 9 per age group). Coomassie blue staining of the membranes used for Western immuno detection was used as loading control. The error bars represent SEM.</p

Analysis of signal transduction in muscle lysates from 11, 29 and 54-week female DU6P and Fzt:DU mice.

<p>Western blot identified phosphorylated and total expression of the respective signaling molecule. Specific activation was calculated from the ratios of phosphorylated versus total protein. Coomassie blue staining of the membranes used for Western immuno detection was used as loading control. A: IGF-1Rß in membrane fractions (n = 3); B: ILK (n = 9); C: GDF-8, 26 kDa band (n = 11) and D: PTEN (n = 9). The inserts provide representative experiments, whereby all samples were studied on the identical membrane. Sample numbers (n) depict the number of samples per age group, the error bars represent SEM.</p

Antibodies used in the study.

<p>Antibodies used in the study.</p

Analysis of protein metabolism in female DU6P and Fzt:DU mice.

<p>A: Western blot of muscle lysates in 11-, 29- and 54-week DU6P and Fzt:DU mice identified different proteins tagged with Ubiquitin for protein degradation (n = 5 per age group). Intensities of the complete lanes were calculated and normalized for the Coomassie blue signals present in the identical complete lane on the membranes used for Western immuno detection. The insert provides a representative experiment, whereby all samples were studied on the identical membrane. Serum levels of 1/3-methyl-histidine (B) and ornithine (C) were analyzed by quantitative HPLC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039711#s2" target="_blank">Materials and Methods</a>. The error bars represent SEM.</p

Analysis of signal transduction in muscle lysates from 11, 29 and 54-week female DU6P and Fzt:DU mice.

<p>Western blot identified phosphorylated and total expression of the respective signaling molecule. Specific activation was calculated from the ratios of phosphorylated versus total protein. A: GSK3ß (n = 9); B: eIF2α (n = 9); C: p70S6 kinase (n = 3); D: ribosomal protein S6 (n = 6). The inserts provide representative experiments, whereby all samples were studied on the identical membrane. Sample numbers (n) depict the number of samples per age group,the error bars represent SEM (n.c.: not calculated due to low signal intensity).</p

Higher levels of muscle glycogen in 29-week female DU6P mice.

<p>Top: Biochemical analysis of glycogen content in muscle tissue of 29-week female Fzt:DU (n = 6) and DU6P (n = 12) mice. Lower: PAS staining of cryosectioned muscle of 29-week Fzt:DU and DU6P female mice. The images correspond to an area of about 1.3×0.88 mm in the histological sections. The error bars represent SEM.</p