Loss of <i>pll</i> promotes dFoxO nuclear localization and transcriptional activity.

<p>(<b>A</b>) Loss of <i>pll</i> does not affect the transcription of <i>dFoxO</i>. Histogram showing the levels of <i>dFoxO</i> mRNAs measured by quantitative RT-PCR. Total RNA of <i>Drosophila</i> third instar larvae were extracted and normalized for cDNA synthesis. Error bars represents standard deviation from three independent experiments. ns stands for not significant. (<b>B-D</b>) Loss of <i>pll</i> promotes nuclear localization of dFoxO. (<b>B</b>) Quantification of the nuclear/cytoplasmic ratio of <i>dFoxO</i>-GFP fusion protein in the fat body shown in <b>C</b> and <b>D</b>. <i>dFoxO</i>-GFP intensities were measured in pixels using Image J. Error bars showed standard deviation from measurement of at least 15 cells for each genotype. Unpaired t test was used to calculate statistical significance, indicated with asterisks (*** <i>P</i><0.001). (<b>C</b> and <b>D</b>) Fluorescence micrographs of fat body cells are shown. Compared with the control (<b>C</b>), loss of <i>pll</i> promotes the translocation of <i>dFoxO</i>-GFP from cytoplasm to nucleus (<b>D</b>). Nuclei were marked with DAPI (blue), cell membranes were stained by anti-Dlg antibody (red). (<b>E</b> and <b>F</b>) X-Gal staining of a <i>Thor</i>-LacZ reporter in third instar larval wing discs. Compared with the control (<b>E</b>), knock-down <i>pll</i> in the wing pouch induced <i>Thor</i> transcription (<b>F</b>). (<b>G</b>) Loss of <i>pll</i> up-regulated the level of <i>Thor</i> mRNA, as measured by quantitative RT-PCR. Total RNA of <i>Drosophila</i> third instar larvae were extracted and normalized for cDNA synthesis. Error bars represented standard deviation from three independent experiments. Unpaired t test was used to calculate statistical significance, indicated with asterisks (* <i>P</i><0.05). Detailed genotypes: (A) Left: <i>act</i>-Gal4/+, Right: <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>act</i>-Gal4/+ (B) Left: <i>Cg</i>-Gal4/+, Right: <i>Cg-</i>Gal4/<i>UAS-pll-IR</i>^<i>V2889</i> (C) <i>Cg-</i>Gal4/<i>+</i>; <i>dFoxO</i>-GFP/+ (D) <i>Cg-</i>Gal4/<i>UAS-pll-IR</i>^<i>V2889</i>; <i>dFoxO</i>-GFP/+ (E) <i>Sd</i>-Gal4/+; <i>Thor</i>-LacZ/+ (F) <i>Sd</i>-Gal4/+; <i>Thor</i>-LacZ/<i>UAS-pll-IR</i>^<i>V2889</i> (G) Left: <i>act</i>-Gal4/+, Right: <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>act</i>-Gal4/+.