Effect of mitochondrial sirtuin expression on glycolysis.

<p>Seahorse extracellular acidification rates (A) were measured in quadruplicate wells containing equal numbers of cells. The experiment was repeated with similar results. Data collected over the first 30 minutes were averaged to yield the basal glycolytic rate (B). Oligomycin-stimulated glycolysis (C) was calculated by subtracting the basal values from the maximum values obtained immediately after oligomycin injection. The oxygen consumption/extracellular acidification ratio (D) was calculated by dividing the values shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106028#pone-0106028-g001" target="_blank">Figure 1D</a> by those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106028#pone-0106028-g003" target="_blank">Figure 3B</a>. All graphs depict means and standard deviations, and *P<0.05. mpH = milli pH units.</p

Sirtuin expression in HEK293 does not affect (A) the rate of cellular growth; (B) mitochondrial mass as judged by western blotting of electron transport chain components; (C)steady-state ATP under basal conditions or after the addition of the metabolic inhibitors etomoxir (Eto, 100 µM), oligomycin (oligo, 1 µM), 2-deoxyglucose (2DG, 100 mM), or combinations thereof; or (D) intramitochondrial NAD⁺.

<p>Growth was measured in quadruplicate wells in two separate experiments which were averaged. ATP was measured in triplicate wells containing equal numbers of cells in two separate experiments which were averaged. NAD+ was measured in three separate preparations of mitochondria and the results averaged. All data are means and standard deviations.</p

Seahorse XF24 extracellular flux analysis of sirtuin-expressing HEK293 cells under 5 mm glucose conditions.

<p>(A) Oxygen consumption and (B) extracellular acidification rates were measured under the same protocol as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106028#pone-0106028-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106028#pone-0106028-g003" target="_blank">3</a>. All graphs depict means and standard deviations. mpH = milli pH units.</p

Analysis of DNA lesions in <i>T. cruzi</i> genome after treatment with H₂O₂.

<p><b>A</b>) Nuclear and mitochondrial dose-responses after exposure to increasing H₂O₂ doses. Cells were treated for 15 min. <b>B</b>) Kinetics of damage and repair of the nuclear and mitochondrial fragments after exposure to 200 µM H₂O₂. Cells were treated for 15 min and allowed to recover for the times indicated. Data are expressed as the mean of two biological experiments. Error bars represent standard error of the mean. Statistical analysis used was unpaired <i>t</i> test. Mitochondrial DNA (<b>○</b>); Nuclear DNA (Δ). ***- P value<0,001; ** - P value<0,01.</p

Mitochondrial function after H₂O₂ treatment.

<p>Analysis of the mitochondrial activity by measuring the basal oxygen consumption rate (OCR). Cells were treated with 0 µM and 200 µM H₂O₂ for 20 min and allowed to recover for 24 hours. Measures were done on the Seahorse Extracellular Flux Analyzer XF24. Results shown are representative of the mean of two independent experiments, performed in replicates of 3–4. The basal level of OXPHOS was calculated by the difference between the mean of rates 1 to 4 and the mean of rates 14 to 16. ** - P value<0,01.</p

Subcellular localization of TcOgg1 in <i>T. cruzi</i>.

<p>CL Brener strain was transfected with pTREX_<i>TcOGG1</i>, resulting in the expression of TcOgg1 fused with GFP (Ogg1-GFP), which enabled the visualization of the protein under confocal microscope. DNA was stained with propidium iodide. Images obtained were analyzed with Zeiss LSM Image Browser software. N: nucleus; K: kinetoplast (mitochondrion); DIC: differential interference contrast. 5 uM scale bars is present in C.</p

8-oxoguanine levels in nuclear and mitochondrial DNA of WT and <i>TcOGG1</i>-overexpressor <i>T. cruzi</i>, with or without H₂O₂ treatment.

<p>Cells were incubated with FITC-avidin, which binds to 8-oxoguanine. Slides containing stained parasites were visualized under a fluorescence microscope and fluorescence intensity was measured with ImageJ program. Graphics were plotted using the average of different experiments and statistical analyses used Mann Whitney test. NT: non-treated; kDNA: kinetoplast (mitochondrion) DNA. *** - P value<0,001.</p

Analysis of DNA lesions in <i>TcOGG1</i>-overexpressor <i>T. cruzi</i>.

<p>Ratio between nuclear and mitochondrial DNA lesions from <i>TcOGG1</i>-overexpressing cells in comparison to control cells, after treatment with 200 µM H₂O₂. Both cell populations were treated for 20 min and allowed to recover for up to 24 hours. Data are expressed as the mean of two biological experiments. Error bars represent standard error of the mean. Statistical analysis used was unpaired <i>t</i> test. ** - P value<0,01; * - P value<0,1.</p

Heterologous complementation assay with FF18733 (WT) and CD138 (<i>ogg1</i>-) yeast.

<p><b>A</b>) Qualitative analysis. Cells were transformed with pYEDP (WT (FF.pYEDP) and o<i>gg1</i>- (CD.pYEDP)) or pYEDP_<i>TcOGG1</i> (only <i>ogg1</i>- (CD.pY_<i>TcOGG1</i>)). Yeasts were grown in plates containing glucose (GLU; without expression of the gene inserted in the vector) or galactose (GAL; expression of <i>TcOGG1</i>, due to galactose promoter), without lysine (selection of Lys⁺ mutants). Letters refer to growth on glucose (A, C and E) or galactose (B, D and F). Numbers refer to different clones. <b>B and C</b>) Quantitative analysis. Mutants obtained in the assay showed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042484#pone-0042484-g002" target="_blank">Fig. 2A</a> were counted, originating <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042484#pone-0042484-g002" target="_blank">Figures 2B–C</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042484#pone-0042484-g002" target="_blank">Fig. 2B</a> shows the results for glucose, whereas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042484#pone-0042484-g002" target="_blank">Fig. 2C</a> displays the results for galactose. The graphics were plotted using median and the statistical analysis used was Kruskal-Wallis test (One way ANOVA). FF.pYEDP (•); CD.pYEDP (▪); CD.pYEDP_<i>TcOGG1</i> (▴). ***- P value<0,001; ** - P value<0,01.</p

Alignments with the predicted <i>TcOGG1</i> products.

<p>Amino acid sequence comparison of the predicted product of the <i>OGG1</i> gene from <i>Trypanosoma cruzi</i> (<i>TcOGG1</i>_B; Tc), <i>Trypanosoma brucei</i> (Tb), <i>Leishmania major</i> (Lm), <i>Arabidopsis thaliana</i> (At), <i>Mus musculus</i> (Mm), <i>Homo sapiens</i> (Hs) and <i>Saccharomyces cerevisiae</i> (Sc). Residues shaded in black indicate identical amino acids. Residues shaded in gray are functionally similar. Residues enclosed by the box belong to the HhH-G/PD motif. Asterisks correspond to the Ogg1 catalytic lysine residue and its auxiliary aspartic acid.</p