Effects of CoQ₁₀ supplementation on oxidative stress induced by Rotenone and t-Butyl hydroperoxide (TBH).

<p>ROS were detected following DCFDA fluorescence in cells treated for 24 hours with 100 nM or 10 uM native CoQ₁₀ or Qter®. ROS were induced by 48 hours treatment with 100 nM Rotenone in T67 (A) and H9c2 cells (B) or by 30 minutes exposure to 100 µM TBH in T67 cells (C) and H9c2 cells (D). Data are the mean ± S.D. of at least three different determinations and are expressed as arbitrary fluorescence units (A.F.U.) normalized on protein content. Protein content was evaluated by Lowry method. Asterisks refer to the statistically significant decrease of ROS production in Rotenone/TBH treated samples supplemented with quinones (n = 5, * p≤0.05); ** p≤0.001).</p

UV Spectra of conjugated dienes.

<p>Membrane lipids were extracted from T67 cells treated for 24 hours with Qter (100 nM and 10 µM) and CoQ₁₀ (10 µM) after 30 minutes exposure to 100 µM TBH. Each spectrum was obtained as a difference spectra between TBH treated and TBH untreated samples. Spectra are normalized on total protein content and are representative of three different sets of experiments.</p

Malondialdehyde (MDA) levels in T67 cells treated with native CoQ₁₀ or Qter.

<p>Cells were pre-treated for 24 hours with native CoQ₁₀ and QTer (100 nM and 10 µM). Panel A shows the MDA levels in the absence of external oxidative stress. Panel B shows the MDA levels after 30 minutes exposure to 100 µM TBH. Data are the mean of two different experimental determinations and are normalized on total protein content.</p

HPLC determination of CoQ10 cellular content.

<p>HPLC determination of CoQ₁₀ cellular content in T67 cells (A) and H9c2 (B) treated for 24 hours with native CoQ₁₀ or Qter® at different concentrations (100 nM and 10 µM). Coenzyme Q₁₀ content was measured also in isolated mitochondria from T67 (C) and H9c2 (D) cells. Data were normalized on total cellular and mitochondrial protein content. Values are means ± S.D., n = 3, * p<0.001 vs. control.</p

Respiratory rates of permeabilized H9c2 and T67 cells treated for 24 hours with 100 nM native CoQ₁₀ or Qter®.

<p>Respirometric analyses were performed in the presence of 5 mM Glutamate/Malate or 12,5 mM Succinate/Glycerol 3-phosphate. Respiratory rates are expressed as nmoles O₂ min⁻¹/10⁶ cells ± S.D. from at least three independent experiments, *<i>p</i><0.05 vs. control. **<i>p</i><0.001 vs. control.</p

Respiratory rates of intact H9c2 and T67 cells treated for 24 hours with 100 nM native CoQ₁₀ or Qter®.

<p>Respirometric analyses were performed under endogenous and uncoupled conditions. The maximal uncoupled respiration was measured in the presence of 500 nM FCCP. Respiratory rates are expressed as nmoles O₂ min⁻¹/10⁶cells ± S.D. from at least three independent experiments. *<i>p</i><0.05 vs. control.</p

Effect of Qter® treatment on ATP, protein content and cell growth in H9c2 cells.

<p>H9c2 cells were treated up to 72 hours with 100 nM Qter® and the ATP content was measured at 24, 48 and 72 hours by HPLC analysis (A). Panel B shows the intracellular ATP content after 24 hours treatment with 100 nM Qter® or native CoQ₁₀, measured using luminescence ATP detection assay. Data are reported as arbitrary luminometric units and normalized on total protein content. (Values are means ± S.D.,n = 5, * p≤0.01 vs control). H9c2 cells treated with 100 nM Qter up to 72 hours were assayed for protein content at 24, 48 and 72 hours. Protein content was evaluated by Lowry method (C), (Values are means ± S.D., n = 5, * p≤0.05 vs. control). Cell growth was assessed by trypan blue exclusion method (D).</p

Analysis of physiological mitochondrial superoxide production using MitoSOX Red.

<p>The representative fluorescence images showed the oxidized MitoSOX fluorescence signal in control H9c2 cells (A) and H9c2 cells following 24 treatment with 100 nM Qter® (B). The fluorescence intensity reported in panel C was quantified by Image J software. Values are presented as means ± SD; <i>n</i> = 20. * p≤0.001.</p

Titration of CoQ₁₀ uptake in H9c2 cells.

<p>H9c2 cells were treated with different concentrations of native CoQ₁₀ or Qter® dissolved in colture medium. After 24 hours cells were carefully washed with PBS and CoQ₁₀ was extracted with exane/ethanol 5∶2 from whole cells and its concentration was determined by HPLC analysis. Data are normalized on total cellular protein content. Values are means ± S.D., n = 3, * p<0.001 vs. native CoQ₁₀ treated samples.</p

UV spectra of oxidized and reduced ubiquinone.

<p>T67 cells were treated for 24 hours with 100 nM Qter or 10 µM native CoQ₁₀, then ubiquinone was immediately extracted, from an equal number of cells, with isopropyl alcohol (for further details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033712#s4" target="_blank">Materials and methods</a>) and the UV spectra were recorded between 320 and 240 nm. The ubiquinone extracted from 100 nM Qter treated sample appears to be completely reduced with a maximum absorption peak at 290 nm, while the ubiquinone extracted from 10 µM native CoQ₁₀ treated sample has a maximum absorption peak shifted towards 275 nm, indicating the presence of the ubiquinone oxidized form. Spectra are representative of three different experiments.</p