Mitochondrial membrane potential during the anoxia-culturing period.

<p>Mitochondrial membrane potential decreases during anoxia for 6h. A) Live cell fluorescence microscopy analysis of primary muscle cells treated with the apoptosis marker JC-1. This dye selectively enters the mitochondria, and changes the colour from red to green as the membrane potential decreases. Red staining demonstrates normal function of mitochondria, while green staining demonstrates loss of mitochondrial membrane potential (indicator of apoptosis). All images were captured using the same settings. Nuclei were stained with Hoechst. Scale bar 20 μM. B) Quantification of images in A using at least four randomly chosen images, demonstrating the relative intensity of green emission compared to red emission (+SEM). Asterisks denote significant differences between control and anoxic conditions (*p<0.05).</p

Cytochrome c localization during the anoxia-culturing period.

<p>Cytochrome c was released from mitochondria during anoxia for 6h. Differentiating cells were fixed (ice-cold ethanol) and immunostained with mouse anti-cytochrome c, followed by Alexa 488-conjugated goat anti-mouse (green) before fluorescence microscopy analysis. Mitochondria were stained using mitotracker (red), nuclei were stained with Hoechst (blue). Scale bar 20 μM. Arrows show release of cytochrome c in close proximity to the mitochondria area.</p

Cytoskeletal changes during anoxia.

<p>A) A representative western blot showing formation of desmin aggregates, and degradation of actin during anoxia (0-6h). The Tubulin protein band was unchanged. Cell lysates were subjected to western blotting using antibodies to tubulin, actin and desmin. B) Anoxia induces cytoskeletal re-organization. Differentiating cells (fixed with 2% PFA) and immunostained with rabbit anti-Desmin and mouse anti-α-tubulin, followed by Alexa 488-conjugatede goat anti-rabbit (green) and Alexa 546-conjugated goat anti-mouse (red) before fluorescence microscopy analysis. Actin was stained using Alexa-488 Phalloidin; nuclei were stained with Hoechst (blue). A reorganization of the cytoskeleton was clearly visible with both desmin, tubulin and actin staining. Scale bar 20 μM.</p

List of primers and probes used for quantitative real-time PCR.

<p>List of primers and probes used for quantitative real-time PCR.</p

Caspase activity measured during the anoxic culturing period.

<p>The activities of caspase 3/7 A) and caspase 9 B) measured at 2h, 4h and 6h were reduced during anoxia, compared with control cells. Asterisks denote significant differences between control and anoxic conditions (*p<0.05) ± SEM. C and D) Differentiated muscle cells were pre-incubated with 20 μM Z-LEHD-FMK (irreversible caspas-9 inhibitor) for 30 min before exposure to anoxic conditions. C) Light microscopy pictures demonstrates morphological changes during anoxia treatment. Arrows indicate rounded up, dead cells. D) Live/dead analysis demonstrate increased number of dead cells during 6 h anoxia, but this is unaffected by the irreversible caspase-9 inhibitor. Fluorescence images, as presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182928#pone.0182928.g003" target="_blank">Fig 3</a>, were quantified using Image J, to obtain percentage of dead cells during anoxia. The graphs represents the average of two independent experiments, quantifying a minimum of nine pictures each (+SEM). Asterisks denote significant differences between control and anoxic conditions (*p<0.05).</p

Percent viable cells after culturing under anoxic conditions.

<p>Primary muscle cells cultured in anoxic conditions triggered by oxyrase (EC-oxyrase) for 0–6 h, followed by cell viability measurements (CellTiter-Glo, Promega). The muscle cells were metabolically active during the whole period. The graph represents the average of three independent experiments (±SEM).</p

Representation of 2D-DIGE gel at Day 14.

<p>Proteins picked for identification are outlined with an arrow and the tagged numbers correspond to the same ones indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125157#pone.0125157.t002" target="_blank">Table 2</a>.</p

Protein interaction network of altered proteins at Day 14.

<p>In the network generated by STRING v.9.1, each node represents a protein and each edge represents an interaction, coloured by evidence type (see STRING website for colour legend). The original graphic output was modified including circles to group the proteins according to their regulation. A green line means upregulation and a red line downregulation.</p

List of identified altered proteins at Day 14 of gestation.

<p>Mol. Mass: Molecular Mass; pI: Isoelectric point; Seq. Cov.: sequence coverage.</p><p>List of identified altered proteins at Day 14 of gestation.</p

Protein interaction network of altered proteins at Day 24.

<p>In the network generated by STRING v.9.1, each node represents a protein and each edge represents an interaction, coloured by evidence type (see STRING website for colour legend).</p