Pelle Modulates dFoxO-Mediated Cell Death in <i>Drosophila</i>

<div><p>Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals. Recent studies also suggest a critical role of IRAKs in tumor development, though the underlying mechanism remains elusive. Pelle is the sole <i>Drosophila</i> IRAK homolog implicated in the conserved Toll pathway that regulates Dorsal/Ventral patterning, innate immune response, muscle development and axon guidance. Here we report a novel function of <i>pll</i> in modulating apoptotic cell death, which is independent of the Toll pathway. We found that loss of <i>pll</i> results in reduced size in wing tissue, which is caused by a reduction in cell number but not cell size. Depletion of <i>pll</i> up-regulates the transcription of pro-apoptotic genes, and triggers caspase activation and cell death. The transcription factor dFoxO is required for loss-of-<i>pll</i> induced cell death. Furthermore, loss of <i>pll</i> activates dFoxO, promotes its translocation from cytoplasm to nucleus, and up-regulates the transcription of its target gene <i>Thor/4E-BP</i>. Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly. This study not only identifies a previously unknown physiological function of <i>pll</i> in cell death, but also shed light on the mechanism of IRAKs in cell survival/death during tumorigenesis.</p></div

DataSheet_1_Health-related quality of life in breast cancer patients in Asia: A meta-analysis and systematic review.doc

ObjectivesThe primary purposes of this meta-analysis and systematic review were to evaluate the health-related quality of life (HRQoL) of Asian breast cancer (BC) patients to understand their holistic HRQoL level and provide medical and nursing recommendations to improve and preserve their quality of life.MethodsA comprehensive literature search was conducted to find cross-sectional studies published in Chinese and English concerning HRQoL in BC patients from the inceptions of databases to 14 March 2022. The databases consulted were PubMed, Web of Science, Embase, Cochrane, PsyclNFO, CINAHL, and CNKI. Literature screening, data extraction, risk bias assessment, and data synthesis were independently carried out by two researchers. The Endnote X9 and Stata 15.0 software programs were used during the meta-analysis process.ResultsOut of the 8,563 studies identified, 23 cross-sectional studies involving 3,839 Asian BC patients were included in this meta-analysis. Two tools, namely, European Organization for the Research and Treatment of Cancer Quality of Life Questionnaire C30 (EORTC QLQ-C30) and Quality of Life Questionnaire Breast Cancer module 23 (EORTC QLQ-BR23)—were used to evaluate the HRQoL of BC patients in Asia. The pooled mean of the global health status of Asian BC patients was 58.34 (95% confidence interval [CI]: 53.66–63.02). According to functional subscales of EORTC QLQ-C30 and EORTC QLQ-BR23, Asian BC patients suffered from the worst emotional functioning (pooled mean=66.38; 95% CI: 59.66–73.11) and sexual enjoyment (pooled mean=49.31; 95% CI: 31.97–63.36). In addition, fatigue (pooled mean=42.17; 95% CI: 34.46–49.88) and being upset by hair loss (pooled mean=48.38; 95% CI: 36.64–60.12) were the most obvious symptoms that Asian BC patients experienced according to the meta-analysis results of the EORTC QLQ-C30 and EORTC QLQ-BR23 symptom subscales.ConclusionAsian BC patients experience a relatively low HRQoL due to the prominent decline in their body functions, as well as the unpleasant experiences caused by their symptoms. It is suggested that timely, appropriate, and targeted intervention should be provided in relation to the physical, psychological, and social aspects of Asian BC patients’ lives to enhance their ability to function, relieve them of adverse symptoms, and improve their overall HRQoL.Systematic Review Registrationhttps://www.crd.york.ac.uk/PROSPERO/, identifier CRD42022321165.</p

Responses of Periphyton to Fe₂O₃ Nanoparticles: A Physiological and Ecological Basis for Defending Nanotoxicity

The toxic effects of nanoparticles on individual organisms have been widely investigated, while few studies have investigated the effects of nanoparticles on ubiquitous multicommunity microbial aggregates. Here, periphyton as a model of microbial aggregates, was employed to investigate the responses of microbial aggregates exposed continuously to Fe₂O₃ nanoparticles (5.0 mg L^–1) for 30 days. The exposure to Fe₂O₃ nanoparticles results in the chlorophyll (a, b, and c) contents of periphyton increasing and the total antioxidant capacity decreasing. The composition of the periphyton markedly changes in the presence of Fe₂O₃ nanoparticles and the species diversity significantly increases. The changes in the periphyton composition and diversity were due to allelochemicals, such as 3-methylpentane, released by members of the periphyton which inhibit their competitors. The functions of the periphyton represented by metabolic capability and contaminant (organic matter, nitrogen, phosphorus and copper) removal were able to acclimate to the Fe₂O₃ nanoparticles exposure via self-regulation of morphology, species composition and diversity. These findings highlight the importance of both physiological and ecological factors in evaluating the long-term responses of microbial aggregates exposed to nanoparticles

<i>dFoxO</i> is required for loss-of-<i>pll</i> induced wing phenotypes.

<p>(<b>A</b>-<b>R</b>) Light micrographs showing <i>Drosophila</i> adult wings, anterior is to the left and distal up. Compared with controls (<b>A</b>, <b>G</b> and <b>M</b>), the wing phenotypes of <i>ptc</i>><i>pll-IR</i>^<i>V2889</i> (<b>B</b>), <i>Sd>pll-IR</i>^<i>V2889</i> (<b>H</b>) and <i>Omb>pll-IR</i>^<i>V2889</i> (<b>N</b>) flies were suppressed by knocking-down <i>dFoxO</i> (<b>D</b>, <b>J</b> and <b>P</b>) or removing one or both copies of <i>dFoxO</i> (<b>E</b>, <b>F</b>, <b>K</b>, <b>L</b>, <b>Q</b> and <b>R</b>), but not by expressing a <i>GFP-IR</i> (<b>C</b>, <b>I</b> and <b>O</b>). In <b>A</b>-<b>F</b>, the lower panels are high magnification of the boxed areas in upper panels. (<b>S</b>-<b>U</b>) Statistical analysis of the ACV phenotype (<b>S</b>) and the adult wing size/wild type (WT) (<b>T</b> and <b>U</b>) as shown in figures <b>A</b>-<b>F</b>, <b>G</b>-<b>L</b> and <b>M</b>-<b>R</b> respectively. One-way ANOVA with Bonferroni multiple comparison test was used to compute <i>P</i>-values, significance is indicated with asterisks (*** <i>P</i><0.001). ns stands for not significant. Detailed genotypes: (A) <i>ptc-</i>Gal4/<i>+</i> (B) <i>ptc-</i>Gal4/<i>UAS-pll-IR</i>^<i>V2889</i> (C) <i>ptc</i>-Gal4/<i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>UAS-GFP-IR</i>/+ (D) <i>ptc</i>-Gal4/<i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>UAS-dFoxO-IR</i>/+ (E) <i>ptc</i>-Gal4/<i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>dFoxO</i>^Δ94/+ (F) <i>ptc</i>-Gal4/<i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>dFoxO</i>^Δ94/Δ94 (G) <i>Sd-</i>Gal4/<i>+</i> (H) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+ (I) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>UAS-GFP-IR</i>/+ (J) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>UAS-dFoxO-IR</i>/+ (K) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>dFoxO</i>^Δ94/+ (L) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>dFoxO</i>^Δ94/Δ94 (M) <i>Omb-</i>Gal4/<i>+</i> (N) <i>Omb-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+ (O) <i>Omb-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>UAS-GFP-IR</i>/+ (P) <i>Omb-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>UAS-dFoxO-IR</i>/+ (Q) <i>Omb-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>dFoxO</i>^Δ94/+ (R) <i>Omb-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>dFoxO</i>^Δ94/Δ94.</p

<i>dFoxO</i> is required for loss-of-<i>pll</i> induced bristle phenotype on notum.

<p>(<b>A-G</b>) Light micrographs showing bristles on the adult notum. Compared with the <i>pnr</i>-Gal4 control (<b>A</b>), RNAi-mediated depletion of <i>pll</i> in notum resulted in reduced bristle number (<b>B</b>), which was suppressed by knocking-down <i>dFoxO</i> (<b>D</b>), deleting one or both copies of endogenous <i>dFoxO</i> (<b>E</b> and <b>F</b>), or expressing Pll (<b>G</b>), but not by expressing a <i>GFP</i> RNAi that served as a negative control (<b>C</b>). The lower panels show high magnification view of the boxed areas in upper panels. (<b>H</b>) Quantification of bristles number in the boxed areas from <b>A-G</b>. One-way ANOVA with Bonferroni multiple comparison test was used to compute <i>P</i>-values, significance is indicated with asterisks (*** <i>P</i><0.001, ** <i>P</i><0.01). ns stands for not significant. Detailed genotypes: (A) <i>pnr-</i>Gal4/<i>+</i> (B) <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>pnr-</i>Gal4/<i>+</i> (C) <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>pnr-</i>Gal4/<i>UAS-GFP-IR</i> (D) <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>pnr-</i>Gal4/<i>UAS-dFoxO-IR</i> (E) <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>pnr-</i>Gal4/<i>dFoxO</i>^{<b><i>Δ94</i></b>} (F) <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>pnr-</i>Gal4 <i>dFoxO</i>^{<b><i>Δ94</i></b>}/<i>dFoxO</i>^{<b><i>Δ94</i></b>} (G) <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>pnr-</i>Gal4/<i>UAS</i>-Pll.</p

Depletion of <i>pll</i> elicits caspases-dependent cell death in adult wing.

<p>(<b>A-D</b>) Light micrographs showing <i>Drosophila</i> adult wings, anterior is to the left and distal up. The loss-of-ACV phenotype in <i>ptc>pll-IR</i>^<i>V2889</i> flies (<b>A</b>) was significantly suppressed by the expression of DIAP1 (<b>C</b>) or Dronc^DN (<b>D</b>), but not that of LacZ (<b>B</b>), which served as a negative control. The lower panels are high magnification of the boxed areas in upper panels (<b>A-D</b>). (<b>E</b>) Quantification of the ACV phenotypes as shown in figures <b>A-D</b>. One-way ANOVA with Bonferroni multiple comparison test was used to compute <i>P</i>-values, significance is indicated with asterisks (*** <i>P</i><0.001). ns stands for not significant. Detailed genotypes: (A) <i>ptc-</i>Gal4/<i>UAS-pll-IR</i>^<i>V2889</i> (B) <i>ptc-</i>Gal4/<i>UAS-pll-IR</i>^<i>V2889</i>; <i>UAS</i>-LacZ/+ (C) <i>ptc-</i>Gal4/<i>UAS-pll-IR</i>^<i>V2889</i>; <i>UAS</i>-DIAP1/+ (D) <i>ptc-</i>Gal4/<i>UAS-pll-IR</i>^<i>V2889</i>; <i>UAS</i>-Dronc^DN/+.</p

Loss of <i>pll</i> produces Toll/NF-κB pathway independent wing phenotype.

<p>(<b>A</b>-<b>L</b>) Light micrographs showing <i>Drosophila</i> adult wings, anterior is to the left and distal up. Compared with <i>ptc</i>-Gal4 control (<b>A</b>), expression of <i>pll</i> RNAi (<i>pll-IR</i>^<i>V2889</i>) induced a loss-of-ACV phenotype (<b>C</b>) that was rescued by expression of Pll (<b>D</b>), while expression of Pll alone showed no obvious defects (<b>E</b>). Expression of RFP was included as a negative control (<b>B</b>). Down-regulation of Toll/NF<b>-</b>κB pathway by expressing RNAi of <i>Toll</i> (<b>F</b>), <i>tube</i> (<b>G</b>), <i>dorsal</i> (<b>I</b>) or <i>Dif</i> (<b>J</b>), or the negative regulator Cactus (<b>H</b>), had no effect on wing development. <i>ptc>pll-IR</i>^<i>V2889</i> was not rescued by co-expressing a <i>cactus</i> RNAi (<b>K</b> and <b>L</b>). The lower panels show high magnification view of the ACV area, boxed in panel A, in upper panels. (<b>M</b>) Quantification of the ACV phenotype as shown in figures <b>A</b>-<b>L</b>. One-way ANOVA with Bonferroni multiple comparison test was used to compute <i>P</i>-values, significance is indicated with asterisks (*** <i>P</i><0.001). ns stands for not significant. Detailed genotypes: (A) <i>ptc</i>-Gal4/+ (B) <i>ptc</i>-Gal4/+; <i>UAS</i>-RFP/+ (C) <i>ptc</i>-Gal4/<i>UAS-pll-IR</i>^<i>V2889</i> (D) <i>ptc</i>-Gal4/<i>UAS-pll-IR</i>^<i>V2889</i>; <i>UAS</i>-Pll; (E) <i>ptc</i>-Gal4/+; <i>UAS</i>-Pll/+ (F) <i>ptc</i>-Gal4/+; <i>UAS-Toll-IR</i>/+ (G) <i>ptc</i>-Gal4/+; <i>UAS-tube-IR</i>/+ (H) <i>ptc</i>-Gal4/<i>UAS</i>-Cactus (I) <i>ptc</i>-Gal4/+; <i>UAS-dorsal-IR</i>/+ (J) <i>ptc</i>-Gal4/+; <i>UAS-Dif-IR</i>/+ (K) <i>ptc</i>-Gal4/+; <i>UAS-cactus-IR</i>/+ (L) <i>ptc</i>-Gal4/<i>UAS-pll-IR</i>^<i>V2889</i>; <i>UAS-cactus-IR</i>/+.</p

<i>pll</i> regulates cell number, but not cell size in adult wing.

<p>(<b>A</b>-<b>D</b>, <b>F</b>-<b>I</b> and <b>K</b>-<b>N</b>) Light micrographs of <i>Drosophila</i> adult wings are shown, anterior is to the left and distal up. Compared with the Gal4 controls (<b>A</b>, <b>F</b> and <b>K</b>), expression of <i>pll</i> RNAi (<i>pll-IR</i>^<i>V2889</i>) driven by <i>Omb</i>-Gal4 (<b>B</b>), <i>Sd</i>-Gal4 (<b>G</b>) or <i>en</i>-Gal4 (<b>L</b>) resulted in reduced wing tissue in the corresponding areas, which were rescued by expression of Pll (<b>D</b>, <b>I</b> and <b>N</b>), but not that of GFP (<b>C</b>, <b>H</b> and <b>M</b>). (<b>E</b> and <b>J</b>) Quantifications of adult wing size/wild type (WT) ratio are shown for figures <b>A</b>-<b>D</b> and <b>F</b>-<b>I</b> respectively (n = 10). One-way ANOVA with Bonferroni multiple comparison test was used to compute <i>P</i>-values, significance is indicated with asterisks (*** <i>P</i><0.001). (<b>O</b> and <b>P</b>) Quantifications of cell size (<b>O</b>) and cell number (<b>P</b>) in wings shown in <b>K</b> and <b>L</b>. The P/A ratio of cell size showed no difference while that of cell number decreased significantly when <i>pll</i> was knocked down in the P compartment by <i>en</i>-Gal4. Unpaired t test was used to calculate statistical significance, indicated with asterisks (*** <i>P</i><0.001, n = 10 in each group). (<b>Q</b>) Statistic analysis of total size P/A ratio are shown for figures <b>K-N</b>. One-way ANOVA with Bonferroni multiple comparison test was used to compute <i>P</i>-values, significance is indicated with asterisks (*** <i>P</i><0.001). ns stands for not significant. Detailed genotypes: (A) <i>Omb-</i>Gal4/<i>+</i> (B) <i>Omb-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+ (C) <i>Omb-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>UAS-</i>GFP/+ (D) <i>Omb-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>UAS-</i>Pll/+ (F) <i>Sd-</i>Gal4/<i>+</i> (G) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+ (H) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>UAS-</i>GFP/+ (I) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>UAS-</i>Pll/+ (K) <i>en</i>-Gal4/+ (L) <i>en</i>-Gal4/<i>UAS-pll-IR</i>^<i>V2889</i> (M) <i>en</i>-Gal4/<i>UAS-pll-IR</i>^<i>V2889</i>; <i>UAS-</i>GFP/+ (N) <i>en</i>-Gal4/<i>UAS-pll-IR</i>^<i>V2889</i>; <i>UAS-</i>Pll/+.</p

Loss of <i>pll</i> elicits apoptotic cell death.

<p>(<b>A-H</b>) AO staining of third instar larval wing discs. Compared with controls (<b>A</b> and <b>E</b>), knock down <i>pll</i> along the A/P boundary by <i>ptc</i>-gal4 (<b>B</b>) or in the wing pouch by <i>Sd</i>-Gal4 (<b>F</b>) triggered cell death in the corresponding areas, which was suppressed by expressing Pll (<b>D</b> and <b>H</b>), but not GFP (<b>C</b> and <b>G</b>). (<b>I-P</b>) X-Gal staining of a <i>hid</i>-LacZ and an <i>rpr</i>-LacZ reporters in wing discs. Compared with controls (<b>I</b> and <b>M</b>), knock down <i>pll</i> in the wing pouch induced <i>hid</i> (<b>J</b>) and <i>rpr</i> (<b>N</b>) transcription, which was blocked by expressing Pll (<b>L</b> and <b>P</b>), but not GFP (<b>K</b> and <b>O</b>). (<b>Q-X</b>) TUNEL staining of third instar larval wing discs. Compared with Gal4 controls (<b>Q</b> and <b>U</b>), knock down <i>pll</i> by <i>Sd</i>-Gal4 (<b>R</b>) or <i>Omb</i>-Gal4 (<b>V</b>) induced cell death in the corresponding areas, which was impeded by expressing Pll (<b>T</b> and <b>X</b>), but not GFP (<b>S</b> and <b>W</b>). In all figures, anterior is to the left and dorsal up. Detailed genotypes: (A) <i>ptc-</i>Gal4/<i>+</i> (B) <i>ptc-</i>Gal4/<i>UAS-pll-IR</i>^<i>V2889</i> (C) <i>ptc-</i>Gal4/<i>UAS-pll-IR</i>^<i>V2889</i>; <i>UAS</i>-GFP/+ (D) <i>ptc-</i>Gal4/<i>UAS-pll-IR</i>^<i>V2889</i>; <i>UAS</i>-Pll/+ (E and Q) <i>Sd-</i>Gal4/<i>+</i> (F and R) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+ (G and S) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>UAS</i>-GFP/+ (H and T) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>UAS</i>-Pll/+ (I) <i>Sd-</i>Gal4/<i>+</i>; <i>hid</i>-LacZ/+ (J) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>hid</i>-LacZ/+ (K) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>hid</i>-LacZ/<i>UAS</i>-GFP (L) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>hid</i>-LacZ/<i>UAS</i>-Pll (M) <i>Sd-</i>Gal4/<i>+</i>; <i>rpr</i>-LacZ/+ (N) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>rpr</i>-LacZ/+ (O) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>rpr</i>-LacZ/<i>UAS</i>-GFP (P) <i>Sd-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>rpr</i>-LacZ/<i>UAS</i>-Pll (U) <i>Omb-</i>Gal4/<i>+</i> (V) <i>Omb-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+ (W) <i>Omb-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>UAS</i>-GFP/+ (X) <i>Omb-</i>Gal4/<i>+</i>; <i>UAS-pll-IR</i>^<i>V2889</i>/+; <i>UAS</i>-Pll/+.</p

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/+.</p