mRNA Levels of <i>sams-1,</i><i>rab-10, drr-1,</i> and <i>drr-2</i> Are Reduced in <i>eat-2(ad1116)</i> Mutants

<p>Relative mRNA levels of <i>sams-1, </i><i>rab-10, drr-1,</i> and <i>drr-2</i> in <i>eat-2(ad1116)</i> compared to N2 were measured by quantitative PCR, and average of four different sample sets are shown. The relative mRNA levels were normalized against the <i>act-1</i> (beta-actin) level in each sample. The RNAi clone for <i>gei-9</i> is shown as a control; this clone does not cause significant lifespan extension when fed to N2 or <i>eat-2</i> worms (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010017#pgen-0010017-st001" target="_blank">Table S1</a>, and data not shown). Error bars: ± SEM.</p

<i>daf-41</i> longevity is dependent on <i>daf-16</i>/FOXO.

<p>(A) <i>daf-16(mgDf50)</i> equally reduced the lifespan of <i>daf-41(ok3052)</i> and N2 worms at 20°C. (B) <i>daf-16(mgDf50)</i> abolished <i>daf-41</i> longevity at 25°C. (C) <i>daf-16(mgDf50)</i> did not further reduce the short life span of <i>daf-41(ok3052)</i> worms at 15°C. (D) Mean lifespan from 3 individual experiments were plotted for the indicated genotypes. Error bars; S.D.; **,p<0.01 versus N2; ††, p<0.01 versus <i>daf-41(ok3052)</i> by t-test. (E) DAF-16 target genes, <i>sod-3</i>, <i>dod-3</i> and <i>lipl-4</i>, were significantly upregulated in response to warm temperature in <i>daf-41(ok3052)</i> relative to N2. n = 4 biological replicates. Error bars, S.E.M; *, p<0.05 versus N2 of 20°C; †, p<0.05 versus <i>daf-41(ok3052)</i> of 20°C by t-test.</p

Ageing data of daf-41 mutants.

<p>Mean, median and maximum lifespan are shown</p><p>*, p<0.05</p><p>**, p<0.01 versus N2,</p><p>^†, p<0.05</p><p>^††, p<0.01 versus daf-41(ok3052),</p><p>^¶, p<0.05</p><p>^¶¶, p<0.01 versus 20°C by t-test.</p><p>Ageing data of daf-41 mutants.</p

<i>daf-41(ok3052)</i> longevity is <i>hsf-1</i> dependent.

<p>(A) <i>hsf-1(sy441)</i> shortened both N2 and <i>daf-41(ok3052)</i> life span at 20°C. (B) <i>hsf-1(sy441)</i> abolished <i>daf-41(ok3052)</i> longevity at 25°C. (C) <i>hsf-1(sy441)</i> further reduced <i>daf-41(ok3052)</i> short life span at 15°C. (D) Mean lifespan from of 3 individual experiments were plotted for indicated genotypes and conditions. Error bars, S.D.; *, p<0.05; **, p<0.01 versus N2; †, p<0.05; ††, p<0.01 versus <i>daf-41(ok3052)</i> by t-test. (E) <i>daf-41(ok3052)</i> enhanced the upregulation of HSF-1 target genes, <i>hsp-16</i>.<i>2</i>, <i>hsp-4</i>, and <i>hsp-70</i>, in response to warm temperature. n = 4 biological replicates. Error bars, S.E.M; *, p<0.05 versus N2 of 20°C; †, p<0.05 versus <i>daf-41(ok3052)</i> of 20°C by t-test. (F) HSF-1 binding activity to HSE was 1.5 fold increased in <i>daf-41(ok3052)</i> at 25°C. Error bars, S.E.M; **, p<0.01 versus N2. (G) At 15°C, <i>daf-21(p673)</i> mutation enhanced the longevity of N2 and <i>daf-41(ok3052)</i>. At 20°C, <i>daf-21</i> mutant animals lived slightly longer than N2. At 25°C, <i>daf-21(p673)</i> animals lived slightly longer than WT but the mutation reduced longevity in the <i>daf-41(ok3052)</i> background.</p

Ageing data of daf-41(ok3052) in various mutant backgrounds.

<p>Mean, median and maximum lifespan are shown</p><p>*, p<0.05</p><p>**, p<0.01 versus N2</p><p>^†, p<0.05;</p><p>^††, p<0.01 versus daf-41(ok3052)</p><p>¶, p<0.05</p><p>^¶¶, p<0.01 versus 20°C by t-test.</p><p>Ageing data of daf-41(ok3052) in various mutant backgrounds.</p

<i>daf-41</i>/ZC395.10 regulates dauer formation and stress resistance.

<p>(A) An alignment of protein sequences between <i>C</i>. <i>elegans</i> DAF-41, <i>D</i>. <i>melanogaster</i> CG16817 and <i>Homo sapiens</i> p23<i>/PTGES3</i>. The similarity between DAF-41 and p23<i>/PTGES3</i> is 44.6%. (B) Schematic illustration of the <i>daf-41</i>, and deletion alleles of <i>ok3015</i> and <i>ok3052</i>. Black arrows indicate the direction of transcription. Red area indicates HSP20-like co-chaperone domain. (C) <i>daf-41</i> mutants constitutively formed dauer larvae (Daf-c) weakly at 25°C and strongly at 27°C. (D) Dauer alae of <i>daf-41(ok3052)</i> animals grown at 27°C are indicated by the white arrows. (E) <i>daf-41(ok3052)</i> worms were resistant for oxidative stress (20mM of H₂O_2, 2.5 hrs) and heat stress (35°C, 8 hrs). <i>gst-4(ok2358)</i> worms were also slightly stress tolerant. (F) <i>daf-41p</i>::<i>gfp</i> (i.e. <i>dpy-5(e907); sEx10796 [rCes daf-41</i>::<i>gfp + pCeh361])</i> worms were labeled with DiI and photos taken at the young adult stage. Patterns of gene expression of <i>daf-41p</i>::<i>gfp</i> (green), DiI (red), and merged figures are shown, with arrows indicating individual neurons.</p

The Dauer-Constitutive Phenotype of <i>daf-2(e1370)</i> Is Enhanced by Many RNAi Clones That Extend Lifespan in a <i>daf-16-</i>Dependent Fashion

<p>Relative dauer formation of <i>daf-2(e1370)</i> animals grown at 22.5 °C on RNAi clones versus vector control is shown, average of two to three experiments. 30–50% of the animals on vector control become dauers at 22.5 °C. Total number of dauers/total number of animals observed is noted on top of bars. Error bars: ± SEM. ‘*', previously characterized RNAi clones [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010017#pgen-0010017-b09" target="_blank">9</a>] served as negative (<i>daf-16,</i> RNAi insert consists of first 1.2 kb cDNA) and positive (<i>daf-2,</i> RNAi insert consists of first 2.2 kb cDNA; see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010017#pgen-0010017-sg002" target="_blank">Figure S2</a>) controls for the dauer experiment. <i>daf-2(e1370)</i> worms grown on <i>ddl-3</i> RNAi gave rise to almost no progeny; therefore, this <i>daf-16</i>-dependent RNAi clone was not assayed.</p

<i>daf-41</i> partially interacts with the chemosensory and thermosensory apparatus to regulate longevity.

<p>(A) Mean lifespan of 3 individual experiments were plotted. The triple mutant of <i>gcy-23(nj37) gcy-8(oy44) gcy-18(nj38) (gcy</i> triple) caused a parallel reduction of lifespan in N2 and <i>daf-41(ok3052)</i>, respectively at 25°C. The <i>gcy</i> triple mutant did not further shorten the life span of daf<i>-41(ok3052)</i> at 15°C. (B) <i>daf-10</i> mutation increased lifespan in parallel to <i>daf-41</i> at 15°C and 20°C. <i>daf-10</i> mutation did not further extend the life span of <i>daf-41(ok3052)</i> worms at 25°C. Error bars, S.D.; *, p<0.05; **, p<0.01 versus N2; †, p<0.05; ††, p<0.01 versus <i>daf-41(ok3052)</i> by t-test. (C) A schematic model describing the regulatory mechanism of longevity by <i>daf-41</i> at different temperatures. At 25°C, <i>daf-41</i> negatively regulates the transcriptional activities of DAF-16 and HSF-1 and their down-regulation results in normal life span. Thermotaxis and steroidal signaling may regulate longevity in parallel to <i>daf-41</i>. At 15°C, <i>daf-41</i> (+) contributes to longevity possibly via <i>daf-16/</i>FOXO. <i>daf-41(+)</i> may also prevent life shortening activities of <i>daf-12(+)</i>, while <i>hsf-1</i> may promote longevity in parallel. These are working models that we interpret with caution, and may reflect direct or indirect interactions.</p

The short life span of <i>daf-41(ok3052)</i> at 15°C is <i>daf-12</i> dependent.

<p>(A-B) <i>daf-12(rh61rh411)</i> reduced longevity of <i>daf-41(ok3052)</i> at 20°C and 25°C. (C) <i>daf-12(rh61rh411)</i> partly rescued the short life span of <i>daf-41(ok3052)</i> at 15°C. (D) Mean lifespan from 3 individual experiments are plotted with indicated genotypes and conditions. Error bars, S.D.; *, p<0.05; **, p<0.01 versus N2; †, p<0.05; ††, p<0.01 versus <i>daf-41(ok3052)</i> by t-test. (E) Transcriptional targets of DAF-12, <i>cdr-6</i> and <i>fard-1</i>, were reduced with temperature in N2, but this tendency was reversed in the <i>daf-41(ok3052)</i> background. n = 4 biological replicates. Error bars, S.E.M; *, p<0.05 versus N2 of 20°C; †, p<0.05 versus <i>daf-41(ok3052)</i> of 20°C by t-test.</p

Genetic interactions of <i>daf-41</i> with dauer signaling pathways.

<p>(A) <i>daf-41(ok3052)</i> Daf-c phenotypes were partially suppressed in <i>af-16(mgDf50)</i> and completely suppressed in <i>daf-12(rh61rh411)</i> backgrounds. (B) <i>daf-41(ok3052)</i> Daf-c phenotypes were suppressed in various chemotaxis mutant backgrounds. (C-D) <i>daf-21(p673)</i> had no additive effect on Daf-c phenotypes at 25°C, and modestly reduced dauer formation at 27°C in the <i>daf-41(ok3052)</i> background. (E) <i>daf-11(m47)</i> had no additive effect on dauer formation in the <i>daf-41(ok3052)</i> background at 22.5°C. (F) <i>hsf-1(sy441)</i> strongly enhanced dauer formation of <i>daf-41(ok3052)</i> at 22.5°C. (G) Cultures of <i>daf-41(ok3052)</i>, <i>hsf-1(sy441) and daf-41;hsf-1</i> are shown grown at 22.5°C. White arrows indicate dauer larvae. All error bars indicate S.D. *, p<0.05; **, p<0.01 versus <i>daf-41(ok3052);</i> ††, p<0.01 versus <i>daf-21(p673)A</i> or <i>hsf-1(sy441)</i> by t-test. (H) <i>daf-41</i> regulates dauer formation via <i>daf-10</i>, <i>daf-12</i> and <i>daf-16</i> similar to <i>daf-21</i>. However, <i>hsf-1</i> suppresses dauer formation in <i>daf-21</i> but not <i>daf-41</i>. Note that the model reflects genetic interactions, not necessarily direct biochemical interactions.</p