Strains used in this study.

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

Tripelennamine strongly inhibits viability and meiotic M-phase in sporulating yeast.

<p>(A) Images of Nomarski microscopy of cells sporulated in the absence of drug (control), in the presence of 2 mM ammonium sulfate (AS), or 100 µM tripelennamine (TA) for 24 hours. Part of the TA image was magnified; arrows indicate granular bodies of unknown origin. (B) 5-fold serial dilution of yeast sporulated for 24 hours in the presence of three different concentrations of TA (indicated on the right) and then transferred to rich media in order to determine the rate of cell survival. Pictures were taken after incubating the rich media agar plates for 2 days at 30°C. (C) FACS analysis of pre-meiotic DNA synthesis of a “no drug” control, and in cells treated with ammonium sulfate (2 mM), or tripelennamine (100 µM). Samples were taken at 2, 3, 4, 6, and 8 hours after induction of sporulation. Staggered histograms show the frequencies (plotted on the y-axis) of relative DNA content, measured as propidium iodide intensity (plotted on the x-axis). (D) Percentage of cells that have completed meiotic M-phase (cells with 2 DAPI foci and cells with 3 or 4 DAPI foci) and those that have formed mature asci over time in sporulation medium (given in hours, x-axis) in the absence (control, circles and solid lines), or the presence of 100 µM tripelennamine (treatment, squares and dashed lines). (E) Expression patterns of representative genes involved in meiotic development (left column), nitrogen catabolite repression (<i>GAT1</i> and <i>GAP1</i>), glycolysis/gluconeogenesis (<i>CDC19</i> and <i>PGK1</i>), and stress response (<i>AZR1</i>). Log2-transformed fluorescence signals of RMA-normalized microarray data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042853#s4" target="_blank">Methods</a>) are plotted on the y-axis and are graphed versus samples taken in rich media and pre-sporulation media (in the absence of tripelennamine), or total time (4 and 8 hours) the cultures spent in sporulation media in the absence (black curve) or presence (red curve) of TA.</p

Tripelennamine sensitizes autophagy-deficient yeast mutants.

<p>(A) Heatmap and dendrogram depicting hierarchical clustering of homozygous deletion pool data from three independent experiments. Quantile normalized and log2-transformed microarray fluorescence signals were analyzed using the Significance Analysis of Microarrays (SAM) software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042853#s4" target="_blank">Methods</a>) and identified 49 genes (indicated on the right-hand side) that were significantly depleted in the presence of tripelennamine (TA) compared to a “no drug” control. Darker shades of red indicate higher fluorescence signals and therefore a higher abundance of that strain in the pool (see legend). (B) Growth of the <i>ho</i>Δ<i>/HO</i> (control) and <i>neo1</i>Δ<i>/NEO1</i> heterozygous deletion strains (see legend) were determined in the presence of various concentrations of (TA) (indicated on the x-axis). Growth rates relative to the “no drug” control (calculated as a ratio of AvgG, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042853#s4" target="_blank">Methods</a>) were determined for each concentration and are plotted on the y-axis. (C) Sporulation efficiency of the <i>ho</i>Δ<i>/HO</i> control strain (black curve) and the <i>neo1</i>Δ<i>/NEO1</i> heterozygous deletion strain (red curve) sporulated in the presence or absence of TA is indicated as percent spores on the y-axis. Both strains were incubated in sporulation media for 48 hours at various concentrations of TA (indicated on the x-axis) and percentage of spores was determined by microscopy. A total of 100 cells were counted for every condition.</p

Small molecule inhibitors of yeast sporulation.

<p>(A) A scatterplot of sensitivity scores (calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042853#s4" target="_blank">Methods</a>) representing the impact of compounds from the NIH clinical collection in the sporulation screening assays. Compounds that inhibit growth or that interfered with the fluorescence-based assay because of auto-fluorescence were not included in this representation. Compounds with a score of greater than 5 are shown in green. DMSO controls are shown in red. (B) A Venn diagram representing the overlap of inhibitors in the growth assay and the two sporulation assays (colored in yellow and green, respectively). 200 compounds had no effect in any of the three assays, 231 inhibited vegetative growth (of BY4741 and/or AD1-9 strains), and 64 inhibited sporulation. The overlap of between growth and sporulation inhibitors was 49. (C) Chemical structures of 12 sporulation-specific inhibitors that were confirmed to inhibit sporulation by microscopy analysis.</p

Two screening assays for sporulation efficiency.

<p>(A) Schematic of the fluorescence-based assay used to identify sporulation inhibitors. Meiotic landmark events indicated as Ent (entry), DS (pre-meiotic DNA replication), Rec (recombination), MI and MII (first and second meiotic division), and Spo (spore formation). The normal final product is depicted as an ascus with four spores (green). Compounds that inhibit sporulation or that are cytotoxic in sporulating yeast cells will suppress the expression of the sporulation-specific gene <i>CDA2</i> and therefore also <i>CDA2</i> promotor-driven <i>GFP</i> expression. (B) Real-time measurement of fluorescence intensities in cells harboring the <i>GFP</i>-reporter, which were sporulated in the presence of varying concentrations of ammonium sulfate (AS) as indicated. Fluorescence of the sporulation culture was measured every 15 min over the course of 20 hours (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042853#s4" target="_blank">Methods</a>). (C) Schematic of the post-recombination growth assay. The two defective alleles (<i>his4x</i> and <i>his4B</i>) can give rise to a functional <i>HIS4</i> allele upon meiotic recombination. The histidine-auxotrophic diploid mother cells can therefore produce histidine-prototrophic spores (indicated in blue). If this event is suppressed by a compound, either because it directly inhibits meiotic recombination or because it is cytotoxic to sporulating yeast cells, no histidine-prototrophs are formed. (D) Proof-of-concept of the <i>his4x</i>/<i>his4B</i> assay. Cells were sporulated for 5 hours in the presence of varying concentrations of ammonium sulfate (AS) as indicated. Aliquots of cells were then transferred to agar plates that lack histidine (-his) or leucine (-leu) and incubated for two days at 30°C, prior to measuring colony density (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042853#s4" target="_blank">Methods</a>) of each spot on the -his agar. Agar plates lacking leucine served as a loading control. The barplot in the upper panel shows mean and standard deviation data from 4 independent experiments.</p

Age-related obesity in NEP-deficient mice.

<p>(<b>a</b>) Comparison of a NEP-knockout (NEP −/−) mouse with an age-matched wild-type animal (NEP +/+). (<b>b</b>) Steatosis of thorax and heart in a NEP-knockout mouse. (<b>c</b>) Abdominal fat accumulation in a NEP-deficient animal. Age-dependent development of body weight in (<b>d</b>) females and (<b>e</b>) males. Data is presented as means ± SEM. Where not shown, error bars lie within the dimensions of the symbols. Average per group 22 mice. Genotype effects ***<i>P</i><0.001 by two-way ANOVA. Significant differences at specific times are calculated by Bonferroni <i>post-hoc</i> test, **<i>P</i><0.01 and ***<i>P</i><0.001. (<b>f</b>) Daily food consumption in 7 and >11 month-old female NEP-knockout (NEP −/−) mice compared with age-matched wild-type animal (NEP +/+), *<i>P</i><0.05 and **<i>P</i><0.01.</p

Effects of candoxatril (Pfizer, UK79,300) on body mass development.

<p>(<b>a</b>) Development of body weight in male C57BL/6 mice starting in an age of 6 months fed with standard diet, supplemented with placebo (solid line and squares) or of the NEP inhibitor candoxatril [consumption 200 mg/kg/day] (broken line and triangles). Treatment day 0 is the first day of treatment. Treatment effect ***<i>P</i><0.001 by two-way ANOVA. Significant differences at specific time-points are calculated by Bonferroni <i>post-hoc</i> test, *<i>P</i><0.05 and **<i>P</i><0.01. (<b>b</b>) Abdominal fat in male C57BL/6 mice after feeding with standard food (open column) or with food supplemented with the NEP inhibitor candoxatril (black column) for two months [consumption 200 mg/kg/day]. Student's <i>t</i>-test **<i>P</i><0.01 versus placebo. (<b>c</b>) Development of food intake in male C57BL/6 mice fed with standard food (open columns) or the same food supplemented with the NEP inhibitor candoxatril (black columns) [consumption 200 mg/kg/day]. Student's <i>t</i>-test **<i>P</i><0.01, ***<i>P</i><0.001 versus placebo. (<b>d</b>) Development of NEP activity in kidney (left panel) and brain (right panel) of mice after oral treatment with candoxatril (1) Placebo, (2) 100 mg/kg/day candoxatril, (3) 200 mg/kg/day candoxatril. Student's <i>t</i>-test *<i>P</i><0.05, **<i>P</i><0.01 versus placebo. (<b>e</b>) Development of body weight in male tumor-bearing mice (Pancreas carcinoma, PSN-1) fed with standard food (solid line and squares) or standard food supplemented with the NEP inhibitor candoxatril (200 mg/kg/day, broken line and triangles). Treatment effect <i>*P<0.05</i>, by two-way ANOVA. Significant differences at specific time-points are calculated by Bonferroni post-hoc test, *<i>P</i><0.05 and **<i>P</i><0.01.</p

Influence of NEP activity on the alcohol-preference ratio in transgenic mice.

<p>(<b>A</b>) No significant differences between NEP-deficient (hatched bars) and NEP wild-type mice (white bars) in absolute alcohol consumption (left panel), total fluid consumption (second left panel), alcohol/total fluid ratio (second right panel), and in alcohol degradation (measured 2 h after alcohol i.p. injection; right panel) under stress-free conditions; (<b>B</b>) Development of stress-induced differences in the preference ratio between the both genotypes; (<b>C</b>) Development of stress-induced differences in the preference ratio between the both genotypes between days 22 and 70 of the experiment; (<b>D</b>) No fading of stress-induced differences after second stress phase in the preference ratio of NEP-deficient and NEP wild-type mice at 3 consecutive periods of approx 30 days. All data sets include at least measurements at 8 independent successive time points. **<i>P</i><0.01, ***<i>P</i><0.001 vs. wild-type.</p

Influence of pharmacological inhibition of NEP activity on the alcohol-preference ratio.

<p>(<b>A</b>) Effects of candoxatril (200 mg/kg/day) on central and peripheral NEP activity; (<b>B</b>) Candoxatril-induced differences in the alcohol preference ratio and (<b>C</b>) total fluid intake. All data sets include at least measurements at 8 independent successive time points. **<i>P</i><0.01, ***<i>P</i><0.001 vs. control.</p

Body composition and diet-depending weight development in NEP-deficient mice.

<p>NMR-monitored influence of feeding on age-dependent body composition in mice fed with low fat diet divided in (<b>a</b>) muscle masses, (<b>b</b>) free body fluid, (<b>c</b>) fat masses, and (<b>d</b>) fat masses per body weight. Effects of (<b>e</b>) low fat and (<b>f</b>) high fat diet on the development of body mass. Data is presented as means ± SEM of at least 10 animals per group. Two-way ANOVA **<i>P</i><0.01; ***<i>P</i><0.001. Where not shown, error bars lie within the dimensions of the symbols. Genotype effects are calculated by two-way ANOVA (**<i>P</i><0.01; ***<i>P</i><0.001). Significant differences at specific time-points are calculated by Bonferroni <i>post-hoc</i> test, *<i>P</i><0.05 and **<i>P</i><0.01.</p