MOESM1 of Pyruvate kinase M2 and the mitochondrial ATPase Inhibitory Factor 1 provide novel biomarkers of dermatomyositis: a metabolic link to oncogenesis

Additional file 1: Figure S1. Linear correlation between the fluorescence intensity and the content of native proteins. HCT116 cell line extracts (0–1 μg/μl) were spotted in the arrays (see Fig. 2a). Significant linear correlations were obtained between the fluorescence intensity (arbitrary units, a.u.) of the spots and the amount of the protein interrogated in the arrays. Protein concentrations in the biopsies were calculated by interpolation in the respective linear plots

MOESM3 of Pyruvate kinase M2 and the mitochondrial ATPase Inhibitory Factor 1 provide novel biomarkers of dermatomyositis: a metabolic link to oncogenesis

Additional file 3: Figure S2. Overexpression of PKM2 in DM. a Tissue extracts (30 µg) derived from two randomly selected muscle biopsies of control donors (CTR), polymyositis (PM), dermatomyositis (DM) and sporadic inclusion body myositis (IBM) and of the HCT116 cell line were fractionated on SDS-PAGE and blotted against anti-PKM2, anti-GAPDH and anti-LDH-A. Electrophoretic migration of the protein is indicated to the left of each blot. b Histograms show the expression of the proteins (a.u.) when compared to the expression in HCT116 cells

MOESM4 of Pyruvate kinase M2 and the mitochondrial ATPase Inhibitory Factor 1 provide novel biomarkers of dermatomyositis: a metabolic link to oncogenesis

Additional file 4: Figure S3. Plasma levels of PKM2 by ELISA and RPPA. a Determination of PKM2 by ELISA in plasma samples of DM, sIBM and CRL patients. b Upper panel, scheme of printing of the plasma samples from CRL, sIBM, DM and PM patients. One nl of 1:20 diluted samples were spotted in quadruplicate. Black boxed: negative controls of BSA; Magenta boxed: standard curves of HCT116 cells; Brown boxed: positive controls of murine IgGs; Orange boxed: standard curve of PKM2 recombinant protein; Green boxed: samples from control donors (CTR); Blue boxed: samples from sIBM patients; Red boxed: samples from DM patients; Yellow boxed: sample from PM patients. Lower panel, parallel array processed with goat anti-mouse IgGs CF647 as negative control. Please note the lack of cross-reactivity against the human plasma IgGs. c The histogram shows the plasma levels of PKM2 quantified by RPPA assay. The results shown are mean ± S.E.M

Identification of metabolites by co-elution with commercial standards.

<p>Extracted ion chromatograms of selected metabolites in plasma samples (purple lines) and commercial standards (blue lines) are shown. The metabolites are defined by their name, ID number, m/z (M+H), retention time, Human Metabolome Data Base ID number and molecular formula.</p

Diagnostic performance of metabolic biomarkers.

<p>ROC curves were plotted to describe performance characteristics of the indicated metabolites in the 57 subject cohort. The Area under the curve (AUC), 95% range of the interval of confidence (IC) and P values are indicated.</p

Scatter plots showing significant correlations between plasma metabolite levels and severity of the disease in CMT1A patients.

<p>Determination of the plasma levels of the metabolites was carried out by UHPLC-MS and its levels correlated with severity of the disease as assessed by CMTNSv2. Healthy controls (blue circles, n = 15), Mild (red squares, n = 15), Moderate (green diamonds, n = 18) and Severe (black triangles, n = 9) groups of CMT1A patients are represented. Metabolites related to (i) protein catabolism (upper row); (ii) mobilization of membrane lipids (middle row) and (iii) muscle biogenesis and the anti-apoptotic function (lower row) are represented. P-value is calculated by analysis of variance; r is the Spearman coefficient.</p

Protein expression in skin biopsies of CMT1A patients.

<p>Protein expression in skin biopsies of CMT1A patients.</p

Analysis of plasma metabolome in CMT1A patients.

<p>(A), Representative UHPLC-MS total ion chromatogram of plasma samples. (B), Plot in a two-dimensional Cartesian coordinate system, with the axes (principal components, PC) representing the greatest variations in the data of Control (blue), Mild (red), Moderate (green) and Severe (black) states related to CMT1A. Three quality control (QC) injections per group are also represented in the plot for the four groups of individuals. 95% confidence ellipses are also included. Triplicate outliers of one of the samples in the Moderate group fall out of the ellipse. (C), Plot of distribution of the plasma samples defined by the two canonical variables (CV1 and CV2) obtained by Canonical Variate Analysis considering the 12 selected metabolites after forward stepwise Linear Discriminant Analysis. The 95% canonical ellipses are also included. Control subjects and mild, moderate and severe CMT1A patients are represented by blue circles, red squares, green diamonds and black triangles, respectively. (D), Histogram showing the content of the 12 metabolites in plasma samples of healthy (blue, n = 15) and CMT1A patients (yellow, n = 42). The results shown are the mean values ± S.E.M. *, P<<i>0</i>.<i>05</i> by <i>Student’s t test</i>.</p

Summary of the major changes in the expression of skin and plasma metabolites during progression of CMT1A.

<p>The loss of muscle and nerves is shown during severity of the disease as revealed by the CMT neuropathy score v2. The changes in biomarkers are illustrated by row thicknesses.</p

SOCE alteration in <i>Gdap1</i>^<i>-/-</i> embryonic motor neurons <b>(A)</b> Fura-2 [Ca²⁺] signals of embryonic MNs from WT (black) and <i>Gdap1</i>^<i>-/-</i> (grey) mice.

<p>After Ca²⁺ release from cell stores with 5 μM thapsigargin (TG) treatment during 7 min in Ca²⁺ free medium, SOCE was activated by adding 2 mM of CaCl₂. Traces were used to obtain <b>(B)</b> maximum Ca²⁺ peak during SOCE and <b>(C)</b> SOCE Ca²⁺ influx (slope). <b>(D)</b> Fura-2 recordings of 5 μM ionomycin elicited [Ca²⁺]_cyt peak in Ca²⁺-free medium. <b>(E)</b> Maximum [Ca²⁺]_cyt peak obtained in Ca²⁺-free medium represents total amount of cytoplasmic Ca²⁺ after cell stores Ca²⁺ release. Traces were obtained averaging at least 100 cells from each genotype. Error bars represent S.E.M. (***p<0.001, Student’s <i>t</i> test).</p