Molecular effects of <i>c.2310_2311dupCC</i> mutation and GP isoforms expression in muscle cells.

<p>(A) Electropherogram of <i>PYGM</i> exon 18 in control C1 (top) and patient P1 (bottom). The square shows the <i>c.2310_2311dupCC</i> mutation. Exonic nucleotides are underlined in black; intronic nucleotides are underlined in grey. Encoded amino acids are indicated (including wild type Arg, and mutant Gly, encoded by codons formed in an exon-exon junction). (B) Immunoblotting showing muscle GP and α-tubulin bands in muscle biopsy homogenates from control C1 and patient P1. (C) Relative contribution of each gene (<i>PYGM</i>, <i>PYGB</i> and <i>PYGL</i>), to the total amount of GP mRNA in undifferentiated and 12 day differentiated cultured muscle cells. Bars represent the result of a single experiment for undifferentiated cells, or mean ± SD of two independent experiments for 12 days differentiated cells. Percentages were calculated as [<i>PYG(x)</i> mRNA x 100/(<i>PYGB</i> mRNA + <i>PYGL</i> mRNA + <i>PYGM</i> mRNA)], using values normalized for the <i>PPIA</i> mRNA. In C1 and C2 skeletal muscle (not shown), <i>PYGB</i> mRNA and <i>PYGL</i> mRNA were negligible (<0.5%).</p

Subjects' information.

<p>*Age at the time the skeletal muscle biopsy was collected. GenBank reference sequence was NP_005600.1. Expression values were calculated considering 100% the mean results obtained in the controls (C1, C2).</p

Immunostaining of myotube cultures with the muscle-specific marker desmin.

<p>Myotubes at 7 days post-differentiation were immunostained with desmin antibody and then cell nuclei were stained with Hoescht. Representative immunofluorescence micrographs of skeletal muscle cultures are shown: (A,B,E,F) C1, (C,D) P1 and (G,H) P2. In A, C, E and G white bars represent 100 µm. In B, D, F and H white bars represent 50 µm.</p

mRNA quantification of GP genes at 0, 7 and 12 days of differentiation.

<p>Results from a single experiment (before differentiation; C1, P1 and P2), or mean of two independent experiments (7-day and 12-day differentiation, C1, C2, P1 and P2) are depicted.</p

Glycogen phosphorylase activity ratio (active/total), glycogen synthase activity ratio (active/total) and glycogen content.

<p>The results represent values for controls (C1, C2) and patients (P1, P2), after 7 days of differentiation. Values are mean ± SEM. The significance of the difference versus controls is: *p<0.01 and **p<0.001. Grey bars represent controls and black bars patients.</p

Immunoblotting analysis for brain/muscle GP, brain GP, liver GP and muscle GS proteins, of 7 days differentiated cell lines.

<p>Grey bars represent controls (C1, C2) and black bars patients (P1, P2). Anti-actin immunoblotting was performed as loading control. Representative images of brain/muscle GP (A), brain GP (B), liver GP (C) and muscle GS (D) are shown. Ratios of intensity of GP (A, B and C) and GS (D) bands compared to intensity of α-actin bands are shown as mean ± SEM. The significance of the difference versus controls is *p<0.05.</p

Residual enzyme activities of mitochondrial respiratory chain complexes in different tissues from the index patient.

<p>Enzyme activities are expressed as</p><p>*cU/U citrate synthase (CS) and</p><p>**nmol.min⁻¹.mg prot⁻¹. CS activity is expressed as mU/mg protein. Abnormal values are indicated in bold. nd, not determined. Complex I, CI; Complex II, CII; Complex III, CIII; Complex IV, CIV.</p

Demographic data and information obtained by low-density array analysis in patients and healthy controls.

<p>PIM, possible intronic mutation.</p>a<p>Age at the time the skeletal muscle biopsy was collected.</p>b<p>GP activity units are µmol/min/g tissue.</p>c<p>Mutations identified in the protein translation product are indicated by a “p” and in the transcript level by a “c” (<a href="http://www.hgvs.org/mutnomen" target="_blank">www.hgvs.org/mutnomen</a>). GenBank reference sequences were NP_005600.1 and NM_005609.1.</p>d<p>Patients's clinical severity was classified based on a numerical scale following the criteria defined by Martinuzzi et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031718#pone.0031718-Martinuzzi1" target="_blank">[17]</a></p><p>Abbreviations: M, male; F, female; NA, data non available; B, biceps; Q, quadriceps; D, deltoid; ND, not determined;</p

Scatter-plots for the six genes presenting the highest differential expression between patients and controls (<i>p</i><0.01).

<p>A. Acetyl-CoA carboxylase beta (<i>ACACB</i>); B. Calpain III large subunit (<i>CAPN3</i>); C. M-cadherin (<i>CADH15</i>); D. Muscle creatine kinase (<i>CKMM</i>); E. Muscle glycogen synthase (<i>GYS1</i>); F. Sarcoplasmic reticulum Ca²⁺ ATPase 1 (<i>SERCA1</i>). Each subject is represented by a dot.</p

BN-PAGE of muscle GP, muscle GS, and SERCA1.

<p>A. BN-PAGE for control C4. In the first dimension (1D), acrylamide percentage ranges from 3% to 12%. In the second dimension (2D), continuous arrows indicate the 2 protein complexes in which muscle GP participates. Discontinuous arrows show muscle GS forming complexes with muscle GP. B. Second dimension of muscle GP BN-PAGE in a control (C7) and patients (P29 and P35).</p