<i>cycG</i>^<i>HR7</i> mutant animals show defects in fat metabolism.

<p>(A) Wild type (Wt) and <i>cycG</i>^<i>HR7</i> mutants similarly ingest food as visualized by blue colored yeast in the larval gut. (A') Survival rate upon wet starvation was measured in <i>cycG</i>^<i>HR7</i> mutant males (red) compared to wild type (Wt, black). Sixfold experiment [n = 15]. (A'') The average survival rate for <i>cycG</i>^<i>HR7</i> is ca 28 hours and for the wild type ca 41 hours, and was taken as the inflexion point of the curve (50% dead animals, arrows in A'). ***p<0.001 according to Student’s T-test. (B-B'') Small lipid droplets are seen in the fat body of a well-fed wild type larva (B, Wt fed). Droplet size increases after of 2 days of amino acid deprivation (B', Wt starved). Similar effects are seen in a well fed <i>cycG</i>^<i>HR7</i> mutant larva (B'', <i>cycG</i>^<i>HR7</i> fed). B-B'' represent Nile Red staining of salivary glands (sg) and the adjacent fat body (fb) and were inverted for better visibility. Scale bar: 50 μm. (C) The weight of third instar larvae of the given genotype is depicted in percent of wild type (Wt). Error bars denote standard deviation [n = 100 per genotype]. ***p<0.001 according to Student’s T-test. (C') Histogram depicting TAG content normalized to protein content of the respective larvae shown in C). Wild type levels (Wt) were taken as 100%. Note increase of TAG content in <i>cycG</i>^<i>HR7</i> mutant larvae by 36% compared to the wild type. Error bars denote standard deviation [n≥3 experiments per genotype]. ***p<0.001 according to Student’s T-test. (D) In contrast to the wild type (Wt, open arrow), <i>cycG</i>^<i>HR7</i> mutant larvae float (marked with arrow) in the buoyancy test. In a hs-<i>CycG</i> background at ambient temperature, <i>cycG</i>^<i>HR7</i> mutant larvae subside to the bottom, as also seen for hs-<i>CycG</i> larvae. (D') Statistical evaluation of the buoyancy test repeated five times with 10 larvae each. Error bars denote standard deviation. ***p<0.001 according to Student’s T-test. (E-J') Oil Red O staining was performed on larval pelts to visualize lipid droplet accumulation in oenocytes. Larvae were either well-fed with yeast paste (E-J), or starved for amino acids (E'-J'). For a statistical evaluation see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005440#pgen.1005440.s003" target="_blank">S3 Fig</a>. The following genotypes were tested: (E-E') wild type (Wt). (F-F') <i>cycG</i>^<i>HR7</i> homozygotes. (G) hs-<i>CycG</i>; <i>cycG</i>^<i>HR7</i> (ambient temperature). (H) hs-<i>CycG</i> (ambient temperature). (I-J') Overexpression of a control (UAS-<i>GFP</i>) or of TSC1/2 specifically in the larval fat body. (I-I') <i>Adh</i>-Gal4/+; UAS-<i>GFP</i>/+ (Adh::GFP). (J-J') <i>Adh</i>-Gal4/+; UAS-<i>TSC1</i> UAS-<i>TSC2</i> (Adh::TSC1/2). Scale bar: 20 μm.