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

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

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

OCR and ECAR in Rat1a cells expressing human Myc point mutants.

<p>(<b>a</b>) A Seahorse Bioscience XF24 Extracellular Flux Analyzer was used for the real-time determination of metabolism. OCR (OXPHOS) was expressed as a function of time. Each inhibitor as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013717#s2" target="_blank">Results</a> was injected at the times indicated by the vertical lines (injections 1–4). A typical experiment, performed in triplicate wells is shown. The experiment was repeated on at least three occasions with similar results. (<b>b</b>) Areas under the curve (AUC) were calculated with the software provided by the manufacturer and used to compare the various cell lines' total reserve respiratory capacity relative to that of vector control cells, which are arbitrarily set at 1. The average of three to five individual experiments, where each cell line was measured at least in triplicate is displayed +/− 1 SE. p-values of the comparisons with vector control cells were calculated by a two-tailed t-test in Microsoft Excel: (*) p≤0.05; (**) 0.01. This graph represents the AUCs for the time point just prior to injection 2 until the time point just before injection 4. This represents the total reserve respiratory capacity of each cell line. (<b>c</b>) ECARs (glycolysis) were expressed as a function of time. The vertical lines represent the same injections as previously described. The experiment was repeated on at least three occasions with similar results. (<b>d</b>) AUCs for the time point just prior to injection 1 to the time point just prior to injection 2 were calculated. This is the best representation of the glycolytic potential of these strains. p-values were calculated and represented as described for (<b>b</b>).</p

Expression of Myc mutants in Rat1a cells.

<p>(<b>a</b>) Diagram of the Myc protein. The approximately 150 residue TRD is shaded, with an expanded MBII domain and relevant amino acids substitutions depicted below the diagram. The basic-helix-loop-helix dimerization domain at the extreme C-terminus of the protein is indicated by the checkered box. (<b>b</b>) Each of the indicated mutations, along with wtMyc was expressed in Rat1a fibroblasts following lentiviral-mediated transduction. An empty lentiviral vector infection served as a negative control. Pooled, blasticidin-resistant colonies were subjected to western analysis for either Myc or β-tubulin, which served as a loading control. The monoclonal antibody used to detect human Myc proteins had some cross reactivity with endogenous rat Myc.</p

Cell death mediated by Myc proteins.

<p>The indicated Rat1a cell lines were seeded into 12 well pates at 10⁵ cells/well and allowed to achieve approximately 80% confluency. At the indicated times, viable cell numbers in triplicate plates were determined by flow cytometry using an Annexin V-Propidium Iodide staining protocol as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013717#s4" target="_blank">Materials and Methods</a>. The results represent the average amount of staining for the triplicate samples +/− 1 SE. The experiment was repeated on at least three occasions with similar results. (<b>a</b>) Serum withdrawal. The cells were washed in PBS and incubated in serum-free medium for the remainder of the study. (<b>b</b>) Glutamine withdrawal. The cells were washed in PBS and incubated in glutamine-free medium for the remainder of the study.</p

Effect of Myc point mutations on proliferation.

<p>The indicated Rat1a cell lines were seeded at 10⁴ cells/well in 12 well plates and allowed to attach in medium containing 10% FBS. At the indicated times, the total cell number in triplicate wells was determined. The points shown represent the average number of cells/well +/− 1 standard error (SE). The experiment was repeated on at least three occasions with similar results. Only adherent cells were counted. The number of cells at the latter time points may be affected by contact inhibition or cell loss due to overgrowth.</p

Effect of Myc point mutations on transformation.

<p>(<b>a</b>) In vitro transformation. Anchorage-independent clonogenic growth in soft agar was assessed as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013717#pone.0013717-Wang1" target="_blank">[54]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013717#pone.0013717-Rothermund1" target="_blank">[56]</a>. 12–14 days after plating, the total number of macroscopically visible colonies on each plate was quantified. Numbers shown represent the average number of colonies seen in triplicate cultures +/− 1 SE. p-values of the comparisons with vector control cells were calculated by a two-tailed t-test in Microsoft Excel: (**) p≤0.01; (***) ≤0.005. (<b>b</b>) In vivo transformation. 5×10⁶ of the indicated Rat1a cell lines were inoculated subcutaneously into the flanks of nude mice. Each inoculum was performed in triplicate and tumor sizes were evaluated weekly. The data shown represents two individually repeated experiments. Averages tumor sizes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013717#pone.0013717-Stone2" target="_blank">[52]</a> +/− 1 SE are depicted as a function of time.</p

¹⁸F-2DG imaging by PET.

<p>Duplicate tumor-bearing mice inoculated with wtMyc, Q131R, or F138C cells were imaged by PET following injection of ¹⁸F-labeled 2DG. Dotted lines indicate tumor boundaries. Arrows indicate the urinary bladder, where the isotope is expectedly the most concentrated. The average relative SUV is indicated with the value for wtMyc set at 1.</p

RNase HII cleavage reaction products generated using various DNA-RNA-DNA hybrid substrates.

<p><b>I</b>; Cartoon of the synthetic substrates used in the <i>in vitro</i> assays, with the sites of incision and expected product size indicated, along with the DNA sequence containing rNMP(s) and mismatched nucleotides. <b>II</b>; The 50-mer duplexes (10 nM) in which the modified strand was 5′ end-labeled (indicated by *), were incubated with Rnase HII for 60 min at 37°C. The DNA duplexes (panel A) as well as DNA-RNA-DNA hybrids containing either single rAMP (panel B), two consecutive rAMPs (panel C), or five rNMPs (panel D) were assayed. The reaction products were separated under denaturing conditions by 15% polyacrylamide gel electrophoresis (PAGE). The efficiency of RNase HII-dependent incision was determined as a percentage of the radioactivity in the incised products relative to the total signal of the substrate (% inc.). The data below the gels are mean values calculated from at least two independent experiments. The DNA sequence containing rNMP(s) is shown alongside the gels where DNA and RNA are represented by uppercase and lowercase letters, respectively. Orange arrows indicate the cleavage sites. The <i>in vitro</i> assays confirm that <i>E.coli</i> RNase HII nicks the DNA backbone 5′ of ribonucleotides embedded in DNA and shows that the efficiency of the reaction is largely unaffected by base-mispairs.</p