<i>aakg-4</i> transcription is directly regulated by DAF-16/FoxO.

<p><b>A, B</b>) <i>aakg-4</i> but not <i>aakg-5</i> mRNA levels are increased in <i>daf-2</i> animals in a <i>daf-16</i> dependent manner. ^♦<i>p</i><0.05, compared to N2, *<i>p</i><0.05, compared to <i>daf-2</i>. Mean values from 4 trials. Error bars, standard deviation. <b>C</b>) DAF-16 binds to the promoter of <i>aakg-4</i> but not <i>aakg-5</i>. <i>aakg-4</i> peak 2 is positioned within 1 Kb of the transcriptional start site and contains two DAF-16 binding elements (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004109#pgen.1004109.s005" target="_blank">Figure S5</a>). One representative experiment is shown which contained 3 immuno-precipitation replicates from the same chromatin preparation (error bars show the standard deviation between them). The horizontal dotted line shows the % input from a negative control region (3′ of gene R11A5.4) that does not bind DAF-16 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004109#pgen.1004109-Schuster1" target="_blank">[22]</a>. Statistical analysis of additional trials is presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004109#pgen.1004109.s021" target="_blank">Table S5</a>. A western blot showing the specificity of the DAF-16 antibody is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004109#pgen.1004109.s004" target="_blank">Figure S4</a>. <b>D</b>) Confocal microscopy shows <i>Paakg-4::gfp</i> to be broadly expressed in <i>C. elegans</i>. Images show <i>Paakg-4::gfp</i> expression pattern in 1 day old hermaphrodites. (<b>i</b>) Whole worm expression pattern. (<b>ii</b>) <i>Paakg-4::gfp</i> is seen in tail sensory organs: phasmid sheath (PhaSh), socket cells (PhaSc) and neurons (PhaN), as well as in epithelial rectal cells (RectE), anal-depressor muscle (ADM), pre-anal ganglion rectal neurons (RectN), body wall muscles (BWM), posterior intestine (Int) and dorsal cord neuronal processes (DC). (<b>iii</b>) <i>Paakg-4::gfp</i> is expressed in vulval (VlvM), uterine muscles (UtM) and ventral cord processes (VC). (<b>iv</b>) Head expression mostly localizes to the ring ganglia (RingN) plus 6 pharyngeal neurons (PhxN). (<b>v</b>) It is also seen in amphid sensory organs including sheath (AmphSh), socket cells (AmphSc) and neurons (AmphN). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004109#pgen.1004109.s026" target="_blank">Table S10</a> compares expression of <i>Paakg-4::gfp</i> with other AMPK subunits. <b>E</b>) Quantification of GFP fluorescence in worms expressing the <i>Paakg-4::gfp</i> reporter shows that fluorescence increased in <i>daf-2</i> animals dependent on <i>daf-16</i>. The same was also true for a second set of strains generated from a different extrachromosomal array. Means from 3 independent trials shown. Error bars, standard error. Animals contained the <i>wuEx256</i> transgene array. ^♦<i>p</i><0.01 compared to N2, *<i>p</i><0.001 compared to <i>daf-2</i>. <b>F</b>) <i>gfp</i> mRNA was increased in <i>daf-2</i> animals in a <i>daf-16</i>-dependent fashion. ^♦<i>p</i><0.05 compared to N2, *<i>p</i><0.01 compared to <i>daf-2</i>. Means from 3 independent trials shown. Error bars, standard deviation. Prior to transgene quantification animals were maintained at 15°C and then shifted to 25°C for 24 hr at the L4 stage.