Prussian blue staining for detection of iron deposits.

<p>The staining was performed on paraffin-embedded sections of 28 days-old flies. No iron deposits were detected in the medulla or retina of either controls or double RNAi flies. Clear iron inclusions were present in paraffin-embedded sections of mouse spleen (positive control).</p

Expression of <i>CG3740</i> and <i>CG11671</i> in heads from 10 days-old flies.

<p>The <i>Ribosomal Protein 49</i> has been used as endogenous control to normalize <i>CG3740</i> and <i>CG11671</i> expression level. (<b>A</b>) <i>CG3740</i> and <i>CG11671</i> have been measured in single RNAi flies (<i>CG3740</i> RNAi and <i>CG11671</i> RNAi) and in double RNAi flies (<i>CG3740</i>; <i>CG11671</i> RNAi) using the same number of males and females (n = 4). The level of <i>CG3740</i> and <i>CG11671</i> in control flies has been set to 1 and the expression of both genes in down-regulated flies expressed relatively. (<b>B</b>) <i>CG3740</i> and <i>CG11671</i> have been measured in single heterozygous deletion flies (<i>Df(1)</i>BSC589/+ and <i>Df(3L)</i>BSC579/+) and in double heterozygous deletion flies (<i>Df(1)</i>BSC589/+; <i>Df(3L)</i>BSC579/+) using female flies (n = 4). The level of <i>CG3740</i> and <i>CG11671</i> in control flies has been set to 1 and the expression of both genes in heterozygous deletion flies expressed relatively. Data refer to the average of three independent experiments ± SD.</p

Alignment of the fly C19orf12 orthologs (CG3740, CG11671) with human C19orf12 protein.

<p>Proteins have been aligned using Clustal 2.1 multiple alignment tool. Stars (*) indicate identities and dots indicate a higher (:) and a lower (.) degree of similarity. The two <i>D. melanogaster</i> proteins are 63% and 55% similar to C19orf12 and share 72% similarity with each other. The transmembrane domains predicted by PolyPhobius are marked in yellow.</p

Detection of vacuoles in ultrathin Epon plastic sections.

<p>A) Horizontal head sections from 28 days-old control (w¹¹¹⁸) and down-regulated (<i>CG3740</i>; <i>CG11671</i> RNAi) flies. B) Number and size of vacuoles have been quantified with Image J running the applications “Find Edges” and “Analyze Particles” on thresholded 8-bit pictures. Particle size was imposed bigger than 20 pixels² and with a circularity factor between 0,5 and 1. Detected vacuoles were displayed using “Overlay Mask”. Data were manually validated to exclude artifacts. Brains analyzed for each fly strain: n = 3. Scale bar: 100 µm.</p

Bang test in young (1 day-old) and aged (1 month-old) control (w¹¹¹⁸ and <i>act</i>-GAL4/+) and double RNAi males.

<p>A) Flies have been vortexed twice (Bang I and II) with 10 minutes in between and the time needed to upright recorded. A) Representative trajectories are reported for aged control (green), double RNAi (red) and for double heterozygous (blue) flies.</p

Expression of <i>CG3740</i> and <i>CG11671</i> in head, thorax and abdomen of <i>D. melanogaster</i>.

<p>The analysis has been performed on total RNA extracted from adult w¹¹¹⁸ flies using the same number of males and females (n = 4). The <i>Ribosomal Protein 49</i> has been used as endogenous control to normalize <i>CG3740</i> and <i>CG11671</i> expression level. The level of <i>CG11671</i> in the abdomen has been set to 1 and the expression of <i>CG3740</i> and <i>CG11671</i> in the other tissues expressed relatively.</p

OCR behavior over time.

<p>(<b>A</b>) Typical time series replicates inside a plate. Behavior of OCR expressed in pmol/min (y-axis) of Fibro_VY_017 over time (x-axis). Colors indicate the row and shape the column of 12-well replicates. Variation increases for larger OCR values, OCR has a systematic well effect, and there are two types of outliers: well-level and single-point. (<b>B</b>) Scatterplot of standard deviation (y-axis) vs. mean (x-axis) of all three time replicates of each interval, well, and plate of OCR of NHDF only shows a positive correlation (<i>n</i> = 409). (<b>C</b>) The same as (B) but for the logarithm of OCR, where the correlation disappears.</p

Statistical testing of M/Ei-fold change patient vs. control on multiple plates.

<p><b>(A)</b> Ratio of M/Ei-fold change (y-axis) of all the cell lines repeated across plates (x-axis) and their respective controls, sorted by the p-value obtained using the OCR-Stats method. Left of the red dashed line are cell lines with significantly lower M/Ei-fold change using OCR-Stats. Dots in orange represent biological samples with significantly lower or higher M/Ei-fold change using the ED method. Highlighted positive (+) and negative (–) controls. <b>(B)</b> Similar to (A), but depicting the p-value in logarithmic scale (y-axis) using OCR-Stats. Red dashed line at <i>P</i> = 0.05. Dots in red represent cell lines with significantly lower M/Ei-fold change using the OCR-Stats method. <b>(C)</b> Quantile-quantile theoretical (x-axis) vs. observed (y-axis) plot of the residuals <i>ϵ</i>_{<i>b</i>,<i>p</i>} of the linear model (2) applied to M/Ei-fold change. Points are lying on the diagonal as expected from normally distributed residuals.</p

Principle of the mitochondrial stress test assay.

<p>(<b>A</b>) Cartoon illustration of OCR levels (y-axis) versus time (x-axis). Injection of the three compounds oligomycin, FCCP, and rotenone delimits four time intervals within each of which OCR is roughly constant. (<b>B</b>) Targets of each compound in the electron transport chain. (<b>C</b>) Typical layout of a mitochondrial stress test 96-well plate.</p

OCR ratios, abbreviations, definitions, metrics, and analogous definitions.

<p>OCR ratios, abbreviations, definitions, metrics, and analogous definitions.</p