When is a slide not a slide? (Or what happens when we think differently about and beyond design)

Primer sequences. (XLS 42 kb

Metabolic interplay of GAPDH and Sir2.

<p>GAPDH catalyzes the oxidation of NADH: it generates NAD⁺ in close proximity to Sir2. Under conditions with high glycolytic flux, higher amounts of glyceraldehyde-3-phosphate (gly-3-P) are produced, driving the backward reaction. In this case, GAPDH reduces more NAD⁺ to NADH and inhibits the deacetylation reaction by depleting NAD⁺; NAD⁺ is a limiting factor for the Sirtuins to catalyze the formation of O-acetyl-ADP-ribose (AADPR) via the transfer of acetyl groups.</p

Overexpression of GAPDH paralogues increases plasmid recombination frequency.

<p>(A) Principle of the <i>lacZ</i>-based plasmid recombination assay (left panel). Plasmid 1 carries a yeast promoter that drives expression of the N-terminal part of ß-galactosidase, and plasmid 2 encodes the C-terminal part. The ß-galactosidase fragments overlap by 564 bp. When the plasmids recombine, a functional <i>lacZ</i> expression vector is reconstituted and yeast colonies turn blue in a membrane-based X-GAL assay. (Right panel) The vectors encoding for both ß-galactosidase fragments were transformed in triplicate with an empty vector, p(<i>HIS3</i>)-<i>SIR2</i>, or p(<i>HIS3</i>)-<i>Eco</i>GAP and were plated on nylon membranes. Cells were grown at 30°C for 4 days, assayed for ß-galactosidase, and air-dried in the dark. Blue and white colonies were counted. Error bars indicate the standard deviation. (B) <i>natMX4</i>-based recombination assay (left panel). Similar to the <i>lacZ</i> assay, two plasmids encoding for overlapping fragments of the <i>natMX4</i> marker cassette were transformed into yeast. When the plasmids recombine, a functional <i>natMX4</i> cassette is reconstituted, allowing growth on NTC- containing media. Yeast containing 5′ and 3′ <i>natMX4</i> plasmids was transformed along with a third plasmid encoding the indicated protein (middle panel). Fifty colonies from each transformation were pooled, grown overnight, and spread on plates in triplicate with or without NTC. The Y axis indicates the number of NTC resistant colonies per 10⁵ cfu. Results are also shown with colonies directly re-spread on the NTC media, without re-growth in liquid media (right panel).</p

Δ<i>tdh1</i>Δ<i>tdh2</i>Δ<i>tdh3</i> yeast survives counter-selection of a GAPDH-encoding plasmid when Sir2 is overexpressed.

<p>(A) The Δ<i>tdh1</i>Δ<i>tdh2</i>Δ<i>tdh3</i> strain MR173 containing the plasmid p(<i>URA3</i>)-<i>Eco</i>GAP was transformed with the indicated <i>HIS3</i> plasmids. Transformants were selected, grown overnight, and spotted in a five-fold serial dilution on SC media containing 5′FOA for counter-selection of the <i>URA3</i> plasmid or on SC media as control. Plates were then incubated for 3 days at 30°C. (B) Similar experiment as described in (A), but using Δ<i>rki1</i> yeast expressing the human <i>RKI1</i> orthologue (Rpi1) from the <i>URA3</i> plasmid. (C) Δ<i>tdh1</i>Δ<i>tdh2</i>Δ<i>tdh3</i> yeast overexpressing Sir2 or its mammalian homologues, human SirT1, human SirT2, and mouse SirT2, were processed as in (A). (D) Similar to (C), but overexpressing the mutant proteins Sir2^H364Y and Sir2^P394L. (E) The yeast strain MR110, in which chromosomal <i>TPI1</i> is deleted and yeast <i>TPI1</i> is expressed from an <i>URA3</i> episome, was transformed with the indicated <i>HIS3</i> plasmids and processed as described above. (F) Similar experiment using the quadruple deletion strain Δ<i>tdh1</i>Δ<i>tdh2</i>Δ<i>tdh3</i>Δ<i>zwf1</i>. (G) Similar to (E), but using yeast strain MR101, which is isogenic to MR110, but expresses human instead of yeast Tpi1 from the <i>URA3</i> episome.</p

Deregulation of the glycolysis/Sir2 equilibrium causes plasmid recombination.

<p>(A) Plasmids isolated from 5′FOA counter-selected, Sir2-overexpressing Δ<i>tpi1</i> and Δ<i>tdh1</i>Δ<i>tdh2</i>Δ<i>tdh3</i> (Δ GAPDH) cells were amplified in <i>E. coli</i> and digested with <i>Eco</i>RI/<i>Sal</i>I (left panel), <i>Kpn</i>I (middle panel) or <i>Bam</i>HI/<i>Xho</i>I (right panel) and compared with equivalently-treated p(<i>HIS3</i>)-<i>SIR2</i>, p(<i>URA3</i>)-yeast <i>TPI1</i>, or p(<i>URA3</i>)-EcoGAP, respectively. (B) p(<i>URA3</i>)-<i>TPI1</i> and the Tpi-encoding plasmid isolated from 5′FOA counter-selected, Sir2-overexpressing Δ<i>tpi1</i> yeast were transformed into BY4741, plated on SC^-HIS and SC^-URA media and incubated at 30°C. (C) Plasmid sequences of p(<i>URA3</i>)-<i>Eco</i>GAP and p(<i>URA3</i>)-yeast Tpi1 were aligned to the sequence of p(<i>HIS3</i>)-<i>SIR2</i>. Identical regions between p(<i>URA3</i>)-<i>Eco</i>GAP and p(<i>HIS3</i>)-<i>SIR2</i> are highlighted in green, regions shared between p(<i>URA3</i>)-yeast <i>TPI1</i> and p(<i>HIS3</i>)-<i>SIR2</i> in blue.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Modeling the individual effect of 100 anti-miRNAs.

<p><b>A and B</b>: Each anti-miRNA is activated when the corresponding miRNA is over-expressed individually (an anti-mir-combination effect is omitted). Each row in this heatmap represents the predicted anti-miRNA effect and the more columns in the same row appears red or orange, the stronger is the predicted effect that this particular miRNA inhibitor can exert on the EGFR signaling pathway. In this manner, we can examine the impact of each anti-miRNA on this signaling pathway.</p

Top 15 anti-miRNAs.

<p>P-value gives the significant level of concentration-differences of all model components between the state of the applied specific anti-miRNA and control state (inactivated anti-miRNA).</p

Histogram of miRNA/target relationships.

<p><b>A</b>: The top ranked 15 miRNAs of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030140#pone-0030140-t002" target="_blank">Table 2</a> correlated with the amount of their corresponding targets in the EGFR signaling pathway (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030140#pone-0030140-t001" target="_blank">Table 1</a>). <b>B</b>: The 15 miRNAs correlated with amount of their corresponding targets in EGFR signaling pathway. The anti-miRNAs of these miRNAs have much less effect on this pathway (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030140#pone-0030140-g005" target="_blank">Fig. 5A and B</a>).</p

Comparison of model predictions with experimental results.

<p><b>A</b>: Experimental results of relative concentration changes of target mRNAs according to individual miRNA over-expression experiments from the Avraham's study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030140#pone.0030140-Avraham1" target="_blank">[30]</a> (Reprinted with permission from AAAS); <b>B</b>: <i>in silico</i> prediction result of relative concentration changes of target-mRNAs according to each miRNA over-expression in the EGFR model. Both heatmaps show very similar qualitative results (protein down-regulation), the only discrepancies are for miR-155 and miR-498. mRNAs with low concentration changes (log2-ratio<0.001) are ignored and shown in ‘white’.</p