HLH-29 Regulates <i>ftn-1</i> Expression.

<p>(A) Schematic representation of the <i>ftn-1</i> transgenes used in this study. Empty triangle, predicted HLH-30 binding site (CACGTG); filled triangle, predicted HLH-29 binding sites (CATGCG or CACGCG) with arrows indicating the corresponding orientations; striped triangle, predicted IDE region containing binding sites for GATA and HRE types of transcription factors. B) Relative intensity of the expression from integrated P_EcoRV<i>ftn-1::GFP</i> in the indicated intestinal rings of wild-type animals (dark bars, N = 61) and <i>hlh-29</i> mutants (light bars, N = 72). C) Relative intensities of the indicated <i>ftn-1</i> reporters in L4 stage, wild type animals subjected to control RNAi (dark bars) or <i>hlh-29</i> RNAi (light bars) RNAi. D) Relative intensities of the indicated <i>ftn-1</i> reporters in L4 stage, wild type animals subjected to control RNAi (dark bars) or <i>hlh-30</i> RNAi (light bars) RNAi. E) RT-qPCR measurements of <i>ftn-1</i> mRNA in wild-type animals and <i>hlh-29</i> mutant subjected to control RNAi (dark bars) or <i>hlh-29</i> RNAi (light bars). F) Relative intensities of the full length <i>ftn-1</i> reporter in L4 stage, <i>hlh-29</i> mutants subjected to control RNAi (dark bars) or <i>hlh-30</i> RNAi (light bars) RNAi. Error bars represent SEM, *P-value <0.05, **P-value <0.005, *** P-value <0.0005).</p

<i>ftn-1</i> mRNA Levels in Adult Stage Animals.

<p>A) RT-qPCR measurements of <i>ftn-1</i> mRNA under normal iron conditions in indicated genetic backgrounds, relative to levels in wild-type animals. B) RT-qPCR measurements of <i>ftn-1</i> mRNA levels in wild-type animals and <i>hlh-29</i> mutants subjected to <i>hif-1</i> RNAi. C) RT-qPCR measurements of <i>ftn-1</i> mRNA in wild-type animals and <i>hlh-29</i> mutants after treatment with BP or FAC. D) RT-qPCR measurements of <i>hlh-29</i> mRNA levels in wild-type animals after treatment with BP or FAC. E) Expression of an integrated transgenic reporter of <i>ftn-1</i> activity in young adult wild-type animals and <i>hlh-29</i> mutants. In panels A - D, error bars represent standard error of the mean (SEM), wild-type = black bars; <i>hlh-29</i> = grey bars; <i>daf-16</i> = striped bars; <i>hlh-29</i>;<i>daf-16</i> = spotted bars. *P-value <0.05, **P-value <0.005, *** -value <0.0005).</p

<i>ftn-1</i> Expression Profile in Wild Type Nematodes.

<p>Stable transgenic lines bearing the EcoRV <i>ftn-1</i> promoter covering 1060 bp upstream and 19 bp downstream of the translation start were examined for expression using fluorescence microscopy as described in Methods. Effects of <i>hlh-29</i> or <i>hlh-30</i> on <i>ftn-1</i> promoter activities were achieved by feeding bacterial strains producing dsRNA for either <i>hlh-29</i> or <i>hlh-30</i> since embryonic stages. Magnified regions emphasize the vulval region in the bright field (BF) images and the posterior intestinal <i>ftn</i>-1 expression in the GFP and merged images.</p

Lifespan Measurements With or Without 24 mM FAC.

<p>Lifespan Measurements With or Without 24 mM FAC.</p

HLH-29 Regulatory Network.

<p>A) Functional annotation and distribution of HLH-29 targets as predicted by DAVID analysis. Brackets indicate the number of target genes in each cluster. B) Protein interaction network of HLH-29 targets that associate with the GO terms “lifespan and aging” (blue ovals), “stress response” (yellow ovals), or “growth” (orange ovals), as predicted by STRING 9.0.</p

Validation of HLH-29 Targets by RT-qPCR.

**<p>RT-qPCR results for these genes were previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059719#pone.0059719-White1" target="_blank">[22]</a>.</p>Δ<p>These genes correlate with previously predicted targets of homodimeric HLH-29 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059719#pone.0059719-Grove1" target="_blank">[48]</a>.</p

t-BOOH Survival.

<p>A) <i>hlh-29</i> mutants show increased resistance to 10 mM t-BOOH when compared to wild-type animals under normal growth conditions or when medium is supplemented with FAC. B) The survival of wild-type animals exposed to 10 mM t-BOOH decreases upon treatment with <i>ftn-1</i> RNAi, while the survival of <i>hlh-29</i> mutants increases. C) <i>ftn-1</i> RNAi does not affect t-BOOH survival in the presence of FAC in wild-type animals, but slightly increases survival of <i>hlh-29</i> mutants. See supplementary tables and methods for statistical information.</p

Schematic Representation of the Regulatory Network for <i>ftn-1</i> Expression.

<p>The previously established regulatory elements are presented with black arrows and the newly established elements from this study are presented with blue arrows. Empty triangle, predicted HLH-30 binding site; filled triangle, predicted HLH-29 binding site; striped triangle, predicted IDE region.</p

Lifespan Assays.

<p>A) The lifespans of wild-type animals and <i>hlh-29</i> mutants are similarly affected by 24 mM FAC. (P-value <0.001). B) <i>ftn-1</i> RNAi increases the lifespan of wild-type animals under normal growth conditions and in the presence of excess iron. C<i>) ftn-1</i> RNAi increases the lifespan of <i>hlh-29</i> mutants grown under normal growth conditions and in the presence of excess iron.</p