Potassium elemental distribution of (parts of) single fibroblast cells obtained from control patient ‘LR’ and Friedreich’s ataxia patient ‘SL’.

<p>For each case, 5 different fibroblasts were scanned. Boundaries of the fibroblasts are indicated, as well as their nuclei (if present). Scans were obtained in ‘Low Dose’ mode, with a step size of 100 nm and a dwell time of 50 ms. Maps are normalized to incoming intensity, 1s measuring time and are dead time corrected.</p

Potassium and iron elemental distribution within control fibroblast case ‘PN’ (upper row) and FRDA fibroblast case ‘DJS’ (lower row).

<p>Scale bar indicates the background-corrected mass fraction in ppm, calculated from a mean cell thickness of 10 μm. Pixel size in the images is 55 nm; acquisition time for each pixel is 50 ms. Elemental maps were measured in ‘High dose’ mode and based on detector no. 5 (XIA05) only. All element maps were normalized to dead time, ring current and quantified using the Fundamental Parameter method, taking into account the ice layer thickness determined using the K-Kα/Kβ ratio. Red striped circles indicate areas with iron hot-spots, circles h1-h3 indicate exogenous iron hot-spots, white arrows spherical structures and red arrows fibre-like structures in the fibroblast cells.</p

Overview of the minor allele frequency (in %) of heteroplasmic variants detected in this study.

<p>Overview of the minor allele frequency (in %) of heteroplasmic variants detected in this study.</p

Nucleotide GC, AC and CT bias plots for the human mtDNA.

<p>The relative coverage as seen in this illustration is based on the average of the relative coverage of the six samples processed with the different protocols: Covaris shearing followed by the Ion Torrent protocol, Covaris shearing followed by the TruSeq procedure and the Nextera XT method. The average relative coverage was calculated for the total relative coverage and for both strand separately.</p

Background-corrected mean mass fractions of the elements P, S, Cl, K, Ca, Mn, Fe, Ni, Cu and Zn in the cytoplasm of 5 fibroblasts from control case ‘LR’ and FRDA case ‘SL’.

<p>Background-corrected mean mass fractions of the elements P, S, Cl, K, Ca, Mn, Fe, Ni, Cu and Zn in the cytoplasm of 5 fibroblasts from control case ‘LR’ and FRDA case ‘SL’.</p

Comparison between the number of variants detected with the MPS and Sanger sequencing technologies.

<p>FN: false negative result, extra: additional low allele frequency variants identified compared to Sanger sequencing.</p>a<p>Ion Torrent PGM results obtained with the TS4.2 software.</p>b<p>MiSeq data obtained with the in-house GATK pipeline.</p><p>Comparison between the number of variants detected with the MPS and Sanger sequencing technologies.</p

Elemental distribution of P, S, Cl, Ca, Zn and Compton scatter within control fibroblast case ‘PN’ (upper row) and FRDA fibroblast case ‘DJS’ (lower row).

<p>Experimental conditions: see legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190495#pone.0190495.g002" target="_blank">Fig 2</a>. White dashed circle indicates the nucleus border. n1-2 indicate the presence of nucleoli.</p

Genome Coverage plots.

<p>Representation of the MPS relative coverage of both strands (rc+: relative coverage of the plus strand, rc-: relative coverage of the negative strand) of the pUC19 plasmid, or mtDNA molecules obtained from the Ion Torrent PGM or MiSeq sequencing system. The outer circle symbolizes the pUC19 (A) or mtDNA (B, C, D) gene structure, respectively. <b>1A:</b> Use of the Ion Torrent PGM standard protocol on the pUC19 plasmid. <b>1B:</b> Use of three different fragmentation methods in combination with the Ion Torrent sequencing protocol on the mtDNA: Ion Shear Plus Reagents (enzymatic), NEBNext dsDNA Fragmentase (enzymatic) and Covaris (physical). <b>1C:</b> Use of an Ion Torrent PGM protocol without PCR amplification in the library construction on the mtDNA. <b>1D:</b> LR-PCR products of the mtDNA were Covaris (physical) or NEBNext dsDNA Fragmentase (enzymatic) sheared, followed by a TruSeq DNA PCR free protocol on a MiSeq instrument. The same six samples were processed with a Nextera XT kit (enzymatic shearing and PCR amplification in library preparation) prior to MiSeq analysis.</p

Prussian blue staining and TEM on FRDA fibroblasts.

<p>a) Light microscopic image of healthy human fibroblasts; b) TEM image of human fibroblast; c) negative Prussian blue staining of human control fibroblasts; d) positive Prussian blue staining of fibroblasts from Friedreich’s ataxia (FRDA) patients. Iron-rich regions are clearly present in the FRDA fibroblast cells as bright blue regions (indicated with arrows), while fibroblast nuclei and cytoplasm have a red and pink color, respectively.</p

Table1.pdf

<p>Aims: Regeneration in skeletal muscle relies on regulated myoblast migration and differentiation, in which the transcription factor nuclear factor of activated T-cells 5 (NFAT5) participates. Impaired muscle regeneration and chronic inflammation are prevalent in myositis. Little is known about the impact of inflammation on NFAT5 localization and expression in this group of diseases. The goal of this study was to investigate NFAT5 physiology in unaffected myoblasts exposed to cytokine or hyperosmolar stress and in myositis.</p><p>Methods: NFAT5 intracellular localization and expression were studied in vitro using a cell culture model of myositis. Myoblasts were exposed to DMEM solutions enriched with pro-inflammatory cytokines IFN-γ with IL-1β or hyperosmolar DMEM obtained by NaCl supplementation. NFAT5 localization was visualized using immunohistochemistry (IHC) and Western blotting (WB) in fractionated cell lysates. NFAT5 expression was assessed by WB and RT-qPCR. In vivo localization and expression of NFAT5 were studied in muscle biopsies of patients diagnosed with polymyositis (n = 6), dermatomyositis (n = 10), inclusion body myositis (n = 11) and were compared to NFAT5 localization and expression in non-myopathic controls (n = 13). Muscle biopsies were studied by means of quantitative IHC and WB of total protein extracts.</p><p>Results: In unaffected myoblasts, hyperosmolar stress ensues in NFAT5 nuclear translocation and increased NFAT5 mRNA and protein expression. In contrast, pro-inflammatory cytokines did not lead to NFAT5 nuclear translocation nor increased expression. Cytokines IL-1β with IFN-γ induced colocalization of NFAT5 with histone deacetylase 6 (HDAC6), involved in cell motility. In muscle biopsies from dermatomyositis and polymyositis patients, NFAT5 colocalized with HDAC6, while in IBM, this was often absent.</p><p>Conclusions: Our data suggest impaired NFAT5 localization and expression in unaffected myoblasts in response to inflammation. This disturbed myogenic NFAT5 physiology could possibly explain deleterious effects on muscle regeneration in myositis.</p