Yki activity does not promote growth by up-regulating InR/TOR signaling.

<p><b>(A–C)</b> Wing discs labelled for phospho-Akt S505, an indicator of InR pathway activity, bearing mutant clones marked black by absence of GFP (green); <i>w</i>.<i>t</i>. twin spots are marked by bright green. (<b>A</b>) <i>pten</i>^<i>1</i> (positive control), (<b>B</b>) <i>ex</i>^<i>e1</i>, and (<b>C</b>) <i>wts</i>^<i>X1</i> (experimentals, having elevated Yki activity owing to reduced or absent phosphorylation by Wts). Phospho-Akt S505 is not increased in (<b>B</b>) and (<b>C</b>), in contrast to (<b>A</b>). <b>(D)</b> Western blot of protein extracts derived from late third instar wing discs of the genotypes shown, labelled for phospho-Akt S505 (Total Akt and β-actin were used as loading controls; <i>pten</i>^<i>1</i>/<i>pten</i>^<i>dj189</i> was used as a positive control; the reduction in S505 staining in the <i>Tsc1</i>^<i>Q87X</i>/<i>Tsc1</i>^<i>PA23</i> lane is due to feedback of TOR activation onto Akt phosphorylation [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002274#pbio.1002274.ref039" target="_blank">39</a>]). In contrast to reduced Pten, loss of either Ex or Wts does not cause an increase in pAkt S505. <b>(E)</b> Blot of same genotypes as in (<b>D</b>), labelled for phospho-S6 Kinase T398, an indicator of TOR pathway activity (β-Tubulin was used as a loading control, and runs as two species). As observed for phospho-Akt S505, Phospho-S6 Kinase T398 levels are elevated by loss of Pten and Tsc activity, but not by loss of either Ex or Wts activity. (<b>F–I</b>): Wing discs from late third instar larvae-bearing MARCM clones expressing <i>UAS</i>.<i>p35</i> (labelled positively with GFP-NLS, green; nuclei are counterstained with Hoechst, blue). The genotypes of clones are (<b>F</b>) <i>UAS</i>.<i>p35</i>, (<b>G</b>) <i>yki</i>^<i>b5</i>+<i>UAS</i>.<i>p35</i>, (<b>H</b>) <i>yki</i>^<i>b5</i>+ <i>UAS</i>.<i>p35+UAS</i>.<i>Dp110</i>, and (<b>I</b>) <i>yki</i>^<i>b5</i><i>+ UAS</i>.<i>p35+UAS</i>.<i>Rheb</i>. <b>J–L</b>: Quantification of clones sizes, cell numbers, and cell sizes from genotypes in F–I. Error bars are Standard Error of the Mean and asterisks denote significances from <i>t</i> tests (* = <i>p</i> < 0.05, ** = <i>p</i> < 0.01, *** = <i>p</i> <0.001, <i>n</i>. <i>s</i>. = not significant). <i>n</i> = 42 (<i>p35</i>), 49 (<i>yki</i>^<i>b5</i><i>+p35</i>), 76 (<i>yki</i>^<i>b5</i><i>+p35+Dp110</i>), 76 (<i>yki</i>^<i>b5</i><i>+p35+Rheb</i>). Expression of either <i>UAS</i>.<i>Dp110</i> or <i>UAS</i>.<i>Rheb</i> results in an increase in the size of <i>yki</i> mutant clones caused by an increase in cell size but not cell number.