A Physicochemical Approach Toward Extending Conjugation and the Ordering of Solution-Processable Semiconducting Polymers

Poly­(3-hexylthiophene)­s (P3HTs) were synthesized with a well-controlled molecular weight (<i>M</i>w) and degree of regioregularity; additionally, π-conjugated P3HT structures in both solutions and films were systematically investigated. Conjugated P3HT phases in spin-cast films significantly changed from ordered nanorods, -fibrils, and -ribbons to less-ordered granules, depending on the conformation of the P3HT chains in solutions. The chain conformations could be physicochemically adjusted by modifying chain lengths (from 5 to 45 kDa), solvents, and ultrasonication. Highly extended conformations of the P3HT in ultrasound-treated solutions yielded longer degree of conjugation both the intra- and intermolecularly. When toluene was used as a marginal solvent, ultrasonicated 0.1 wt % 29 kDa P3HT solutions could be used to yield highly ordered aggregates in spin-cast films, including nanoribbons or nanosheets, with field-effect mobility (μ_FET) up to ∼0.1 cm² V^–1 s^–1 being measured for organic field-effect transistors (OFETs). However, ultrasonicated chloroform systems with good P3HT solubility (for P3HT <i>M</i>w ≥ 20 kDa) yielded featureless conducting layers even at 0.4 wt % P3HT content. However, these film-based OFETs yielded μ_FET values up to 0.04 cm² V^–1 s^–1, which were much greater than 0.004 cm² V^–1 s^–1 for the nonultrasonicated systems

Temperature-Driven Phase Transition of a Fused Dithienobenzothiadiazole–Tetrathiophene Based Semiconducting Copolymer

A high-molecular-weight donor–acceptor (D–A) copolymer, pDTfBT-4T, poly­(5,8-di­[3-octyl­dodecan­thiopen-5-yl]­dithieno­[3′,2′:3,4:2″,3″:5,6]­benzo­[1,2-<i>c</i>]­[1,2,5]-thiadiazole-<i>alt</i>-2,2′-bithiophene), including alternating dithieno­[3′,2′:3,4;2″,3″:5,6]­benzo­[1,2-<i>c</i>]­[1,2,5]­thiadiazole (DTfBT) and 3-octyl­dodecyl­tetrathiophene (4T) was synthesized by a Stille coupling polymerization. We found that pDTfBT-4T had a molecular number-average weight of 276 kg mol^–1 and formed unexpectedly strong interchain aggregates in dilute solutions at room temperature, which was similar to those in as-spun thin solid films. pDTfBT-4T thin films were spun-cast from a warm dilute chlorobenzene solution on hydrophobic polymer-treated SiO₂ dielectrics. Some were shortly annealed at various temperatures (<i>T</i>) for 10 min to improve π-overlapped structures as charge-carrier transport paths. The ordered domains of pDTfBT-4T in the annealed films could be tuned from one-dimensional nanorods to two-dimensional nanosheets with an increasing in <i>T</i>, which also provided enhanced crystal orientation. Optimizing the conjugated structures of pDTfBT-4T in the annealed films, the polymer based OFETs yielded a hole mobility up to 1.45 cm² V^–1 s^–1 as well as negligible hysteresis and excellent negative bias stability

Engineered Split-TET2 Enzyme for Inducible Epigenetic Remodeling

The Ten-eleven translocation (TET) family of 5-methylcytosine (5mC) dioxygenases catalyze the conversion of 5mC into 5-hydroxymethylcytosine (5hmC) and further oxidized species to promote active DNA demethylation. Here we engineered a split-TET2 enzyme to enable temporal control of 5mC oxidation and subsequent remodeling of epigenetic states in mammalian cells. We further demonstrate the use of this chemically inducible system to dissect the correlation between DNA hydroxymethylation and chromatin accessibility in the mammalian genome. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype–phenotype relations in various biological systems

<i>Drosophila</i> DJ-1 Decreases Neural Sensitivity to Stress by Negatively Regulating Daxx-Like Protein through dFOXO

<div><p><i>DJ-1</i>, a Parkinson's disease (PD)–associated gene, has been shown to protect against oxidative stress in <i>Drosophila</i>. However, the molecular mechanism underlying oxidative stress-induced phenotypes, including apoptosis, locomotive defects, and lethality, in <i>DJ-1</i>-deficient flies is not fully understood. Here we showed that <i>Daxx-like protein</i> (<i>DLP</i>), a <i>Drosophila</i> homologue of the mammalian <i>Death domain-associated protein</i> (<i>Daxx</i>), was upregulated under oxidative stress conditions in the loss-of-function mutants of <i>Drosophila DJ-1β</i>, a <i>Drosophila</i> homologue of <i>DJ-1</i>. <i>DLP</i> overexpression induced apoptosis via the c-Jun N-terminal kinase (JNK)/<i>Drosophila</i> forkhead box subgroup O (dFOXO) pathway, whereas loss of <i>DLP</i> increased resistance to oxidative stress and UV irradiation. Moreover, the oxidative stress-induced phenotypes of <i>DJ-1β</i> mutants were dramatically rescued by <i>DLP</i> deficiency, suggesting that enhanced expression of <i>DLP</i> contributes to the <i>DJ-1β</i> mutant phenotypes. Interestingly, we found that dFOXO was required for the increase in <i>DLP</i> expression in <i>DJ-1β</i> mutants and that dFOXO activity was increased in the heads of <i>DJ-1β</i> mutants. In addition, subcellular localization of DLP appeared to be influenced by <i>DJ-1</i> expression so that cytosolic DLP was increased in <i>DJ-1β</i> mutants. Similarly, in mammalian cells, Daxx translocation from the nucleus to the cytosol was suppressed by overexpressed <i>DJ-1β</i> under oxidative stress conditions; and, furthermore, targeted expression of <i>DJ-1β</i> to mitochondria efficiently inhibited the Daxx translocation. Taken together, our findings demonstrate that DJ-1β protects flies against oxidative stress- and UV-induced apoptosis by regulating the subcellular localization and gene expression of DLP, thus implying that Daxx-induced apoptosis is involved in the pathogenesis of <i>DJ-1</i>-associated PD.</p> </div

<i>DLP</i> deficiency reduces acute sensitivity to oxidative stress and UV, and improves locomotive dysfunction in <i>DJ-1β</i> mutant.

<p>(A) Comparison of the survival rates of <i>DLP</i> and <i>DJ-1β</i> double mutants (<i>DLP¹</i>; <i>DJ-1β^ex54</i> and <i>DLP²</i>; <i>DJ-1β^ex54</i>) with wild-type (WT) and <i>DJ-1β^ex54</i> flies under oxidative stress conditions (log-rank test: WT, n = 250; <i>DJ-1β^ex54</i>, n = 250; <i>DLP¹</i>; <i>DJ-1β^ex54</i>, n = 250; <i>DLP²</i>; <i>DJ-1β^ex54</i>, n = 250, p<0.01, groups with the same letter do not differ significantly). (B) Reduced oxidative stress-induced apoptosis was noted in the larval brain of the <i>DLP</i> and <i>DJ-1β</i> double mutant (<i>DLP¹</i>; <i>DJ-1β^ex54</i>) compared to <i>DJ-1β^ex54</i>. The larval brains were treated with 0.1% H₂O₂ for 24 h and cell death was detected via acridine orange staining. (C) Sensitized DA neuronal death of <i>DJ-1β^ex54</i> under oxidative stress conditions was rescued by <i>DLP</i> deficiency. The flies were fed with 1% H₂O₂ for 3 days. (n = 10, Student's <i>t</i>-test: DM, ** p<0.01, *** p<0.001; DL1, ** p<0.01; PM, ** p<0.01). (D) Survival rates of WT, <i>DJ-1β^ex54</i>, and double mutant of <i>DLP</i> and <i>DJ-1β</i> (<i>DLP¹</i>; <i>DJ-1β^ex54</i> and <i>DLP²</i>; <i>DJ-1β^ex54</i>) pupae after exposure to UV irradiation (10 mJ/cm²; black bars) as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003412#s4" target="_blank">Materials and Methods</a> (Kruskal-Wallis test: CTL, n≥6, p>0.1; UV, n≥5, p<0.01, groups with the same letter do not differ significantly). CTL, UV-untreated control pupae; UV, UV-treated pupae. (E) Comparison of climbing abilities of WT, <i>DLP¹</i>, <i>DJ-1β^ex54</i>, and double mutants of <i>DLP</i> and <i>DJ-1β</i> (<i>DLP¹</i>; <i>DJ-1β^ex54</i>). The climbing abilities of 5-day-old flies for each group were tested as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003412#s4" target="_blank">Materials and Methods</a> (ANOVA and Tukey's HSD analysis: n≥12, p<0.01, groups with the same letter do not differ significantly). All data are expressed as means ± s.e. values.</p

Schematic representation of the role of <i>Drosophila</i> DJ-1 in the cellular response to oxidative stress or UV.

<p>dFOXO, DLP, and JNK form a circuit that controls cellular responses to stress, and DJ-1 sets neural sensitivity to stress by regulating this circuit.</p

Decreased DA neurons and increased apoptosis in <i>DJ-1β</i> mutant under oxidative stress conditions.

<p>(A) DA neurons visualized by immunohistochemical analysis with anti-tyrosine hydroxylase antibody in the brains of wild-type (WT) and <i>DJ-1β</i> mutant (<i>DJ-1β^ex54</i>) flies fed with 1% H₂O₂ for 3 days. Dotted boxed areas indicate DA neuron clusters. The lower pictures, including DM, DL1, DL2, and PM, are the magnified 4 dotted boxed areas of the upper pictures. Magnification of the upper pictures, 100×; Magnification of the lower pictures, 400×. (B) Graphs showing the number of DA neurons in each cluster of WT and <i>DJ-1β^ex54</i> flies after feeding with H₂O₂ for 3 days (n = 10, Student's <i>t</i>-test: DM, *** p<0.001; DL1 and PM, ** p<0.01). The data are expressed as means ± s.e. values. (C) Acridine orange staining of 0.1% hydrogen peroxide-treated larval brains showed that increased oxidative stress-induced apoptosis in <i>DJ-1β^ex54</i> compared to the WT controls. DM, dorsomedial clusters; DL, dorsolateral clusters; PM, posteriomedial clusters.</p

DLP activates apoptosis and the JNK/dFOXO signaling pathway.

<p>(A) Comparison of tissue sizes of control (<i>MS1096</i>/Y), <i>DLP</i>-overexpressing (<i>MS1096</i>><i>DLP</i> and <i>MS1096</i>><i>DLP</i>^×<i>2</i>), and <i>DLP</i>- and <i>DIAP1</i>-coexpressing (<i>MS1096</i>><i>DLP</i>^×<i>2</i>+<i>DIAP1</i>) fly wings. Two copies of the <i>DLP</i> gene were overexpressed in <i>MS1096</i>><i>DLP</i>^×<i>2</i>. (B) Acridine orange-stained images of control (<i>MS1096</i>/Y), <i>DLP</i>-overexpressing (<i>MS1096</i>><i>DLP</i> and <i>MS1096</i>><i>DLP</i>^×<i>2</i>), <i>DLP</i>- and <i>hep</i>-coexpressing (<i>MS1096</i>><i>DLP</i>+<i>hep</i>), <i>DLP</i>-overexpressing and <i>hep</i> deficient (<i>MS1096</i>><i>DLP</i>, <i>hep¹</i>), <i>DLP</i>-overexpressing and <i>bsk</i> deficient (<i>MS1096</i>><i>DLP</i>, <i>bsk¹</i>), <i>DLP</i>- and <i>puc</i>-coexpressing (<i>MS1096</i>><i>DLP</i>^×<i>2</i>+<i>puc</i>), and <i>DLP</i>-overexpressing and <i>dFOXO</i> deficient (<i>MS1096</i>><i>DLP</i>^×<i>2</i>, <i>dFOXO²¹</i>) wing imaginal discs. (C) Genetic interactions of <i>DLP</i> with <i>bsk</i>, <i>hep</i>, and <i>puc</i> in the developing wing. The reduced wing phenotype induced by <i>DLP</i> overexpression (<i>MS1096</i>><i>DLP</i>) was strongly exacerbated by <i>bsk</i> (<i>MS1096</i>><i>DLP</i>+<i>bsk</i>) or <i>hep</i> (<i>MS1096</i>><i>DLP</i>+<i>hep</i>) overexpression, and suppressed by <i>bsk</i> (<i>MS1096</i>><i>DLP</i>, <i>bsk¹</i>) or <i>hep</i> (<i>MS1096</i>><i>DLP</i>, <i>hep¹</i>) deficiency or co-expression of <i>puc</i> (<i>MS1096</i>><i>DLP</i>^×<i>2</i>+<i>puc</i>). (D) Comparison of JNK activity in the <i>DLP</i>- and <i>bsk</i>-coexpressing or <i>DLP</i>- and <i>hep</i>-coexpressing wing imaginal discs (<i>MS1096</i>><i>DLP</i>+<i>bsk</i> or <i>MS1096</i>><i>DLP</i>+<i>hep</i>) with <i>DLP</i>-, <i>bsk</i>- or <i>hep</i>-overexpressing wings (<i>MS1096</i>><i>bsk</i> or <i>MS1096</i>><i>hep</i>) by anti-phospho-JNK antibody staining. (E) Genetic interactions of <i>DLP</i> with <i>dFOXO</i> in the developing wing. The wing phenotype of <i>DLP</i> overexpression (<i>MS1096</i>><i>DLP</i>^×<i>2</i>) was strongly suppressed by <i>dFOXO</i> deficiency (<i>MS1096</i>><i>DLP</i>^×<i>2</i>, <i>dFOXO²¹</i>). <i>MS1096</i> with <i>dFOXO</i> deficiency (<i>MS1096</i>, <i>dFOXO²¹</i>) was used as controls. (F) DLP protein levels in the control (<i>sev-GAL4</i>) and constitutive active <i>hep</i>-overexpressing (<i>sev</i>><i>hep^CA</i>) fly heads (Student's <i>t</i>-test, n = 9, *** p<0.001). <i>sev</i>, <i>sevenless-GAL4</i>. The data are expressed as means ± s.e. values. The genotypes of the samples were <i>MS1096</i>/Y (<i>MS1096-GAL4</i>/Y), <i>MS1096</i>><i>bsk</i> (<i>MS1096-GAL4</i>/Y; <i>UAS-bsk</i>/+), <i>MS1096</i>><i>hep</i> (<i>MS1096-GAL4</i>/Y; <i>UAS-hep</i>/+), <i>MS1096</i>, <i>bsk¹</i> (<i>MS1096-GAL4</i>/Y; <i>bsk¹</i>/+), <i>MS1096</i>, <i>hep¹</i> (<i>MS1096-GAL4</i>/<i>hep¹</i>), <i>MS1096</i>><i>DLP</i> (<i>MS1096-GAL4</i>/Y; <i>EY09290</i>/+), <i>MS1096</i>><i>DLP</i>+<i>bsk</i> (<i>MS1096-GAL4</i>/Y; <i>EY09290</i>/<i>UAS-bsk</i>), <i>MS1096</i>><i>DLP</i>+<i>hep</i> (<i>MS1096-GAL4</i>/Y; <i>EY09290</i>/<i>UAS-hep</i>), <i>MS1096</i>><i>DLP</i>, <i>bsk¹</i> (<i>MS1096-GAL4</i>/Y; <i>EY09290</i>/<i>bsk¹</i>), <i>MS1096</i>><i>DLP</i>, <i>hep¹</i> (<i>MS1096-GAL4</i>/<i>hep¹</i>; <i>EY09290</i>/+), <i>MS1096</i>><i>DLP</i>^×<i>2</i> (<i>MS1096-GAL4</i>/Y; <i>EY09290</i>/<i>EY09290</i>), <i>MS1096</i>><i>puc</i> (<i>MS1096-GAL4</i>/Y; <i>UAS-puc</i>/+), <i>MS1096</i>><i>DLP</i>^×<i>2</i>+<i>puc</i> (<i>MS1096-GAL4</i>/Y; <i>EY09290</i>/<i>EY09290</i>; <i>UAS-puc</i>/+), <i>MS1096</i>, <i>dFOXO²¹</i> (<i>MS1096-GAL4</i>/Y;; <i>dFOXO²¹</i>/+), <i>MS1096</i>><i>DLP</i>^×<i>2</i>, <i>dFOXO²¹</i> (<i>MS1096-GAL4</i>/Y; <i>EY09290</i>/<i>EY09290</i>; <i>dFOXO²¹</i>/+), MS1096><i>DLP</i>^×<i>2</i>+<i>DIAP1</i> (<i>MS1096-GAL4</i>/Y; <i>EY09290</i>/<i>EY09290</i>; <i>UAS-DIAP1</i>/+), <i>sev-GAL4</i> (<i>sev-GAL4</i>/+), and <i>sev</i>><i>hep^CA</i> (<i>sev-GAL4</i>/+; <i>UAS-hep^CA</i>/+). <i>bsk</i>, <i>basket</i>; <i>DIAP1</i>, <i>Drosophila inhibitor of apoptosis protein 1</i>; <i>hep</i>, <i>hemipterous</i>; pJNK, phospho-JNK; <i>puc</i>, <i>puckered</i>.</p

Generation and characterization of <i>DLP</i> mutants.

<p>(A) Genomic structure of the <i>DLP</i> gene. Exons of the <i>DLP</i> gene are shown in black (coding region) and white (non-coding region) boxes. The inverted triangles indicate the P-elements, <i>EY09290</i> and <i>KG01694</i>. The deletion sites of <i>DLP¹</i>, <i>DLP²</i>, <i>DLP³</i>, and <i>DLP⁴</i> are illustrated under the genomic structures. (B) Determination of the deleted size in <i>DLP</i> mutants by genomic DNA PCR. (C) Western blotting of DLP in wild type (WT), <i>DLP</i> loss-of-function (<i>DLP¹</i> and <i>DLP²</i>) and gain-of-function (<i>elav</i>><i>DLP</i>) mutants. Intact DLP protein is not detected in the <i>DLP</i> mutants. (D) Comparison of <i>DLP</i> gene expression in the third-instar larval brains of WT and a <i>DLP</i> mutant via RNA <i>in situ</i> hybridization. (E–F) Survival rates of <i>DLP</i> loss-of-function (<i>DLP¹</i>, <i>DLP²</i>, and <i>elav</i>><i>DLPi</i>) and gain-of-function (<i>elav</i>><i>DLP</i>) mutants under oxidative stress conditions. (E) WT and <i>DLP^rv/DLP¹</i> were used as controls (log-rank test: WT, n = 300; <i>DLP¹</i>, n = 250; <i>DLP²</i>, n = 250; <i>DLP^rv/DLP¹</i>, n = 300, p<0.01, groups with the same letter do not differ significantly). (F) <i>elav</i>/Y was used as a control (log-rank test: <i>elav</i>/Y, n = 350; <i>elav</i>><i>DLP</i>, n = 300; <i>elav</i>><i>DLPi</i>, n = 300, p<0.01, groups with the different letter differ significantly). The genotypes of the samples were <i>elav</i>/Y (<i>elav-GAL4</i>/Y), <i>elav</i>><i>DLP</i> (<i>elav-GAL4</i>/Y; <i>EY09290</i>/+), and <i>elav</i>><i>DLPi</i> (<i>elav-GAL4</i>/Y; <i>UAS</i>-<i>DLP-RNAi</i>/+). (G) Acridine orange staining of larval brains of <i>DLP¹</i> and WT treated with 0.1% H₂O₂ for 24 h. (H) Survival rates of WT and <i>DLP</i> mutant (<i>DLP¹</i> and <i>DLP²</i>) pupae after exposure to UV irradiation (10 mJ/cm²; black bars) as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003412#s4" target="_blank">Materials and Methods</a> (Kruskal-Wallis test: CTL, n≥6, p<0.1; UV, n = 6, p<0.01, groups with the same letter do not differ significantly). CTL, UV-untreated control pupae; UV, UV-treated pupae. All data are expressed as means ± s.e. values. (I) TUNEL-stained images of UV-exposed 0–3 h embryos of WT and <i>DLP¹</i>. The lower panels are higher-magnification images of the boxes indicated with dotted lines in the upper panels. CTL, control; rv, revertant; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling.</p

The role of <i>dFOXO</i> in the regulation of <i>DLP</i> by <i>DJ-1β</i>.

<p>(A) DLP protein levels in the heads of control (<i>tub</i>-<i>GAL4</i>), <i>DJ-1β</i> mutant (<i>tub</i>-<i>GAL4</i>, <i>DJ-1β^ex54</i>) and the double mutant of <i>cncC</i> and <i>DJ-1β</i> (<i>tub</i>><i>cncCi</i>, <i>DJ-1β^ex54</i>) flies fed with 1% H₂O₂ (Student's <i>t</i>-test, n = 4, * p<0.05). NS, not significant. (B–C) DLP mRNA (B) and protein (C) levels in the head of WT, <i>DJ-1β^ex54</i>, and <i>dFOXO</i> and <i>DJ-1β</i> double mutant (<i>dFOXO²¹</i>, <i>DJ-1β^ex54</i>) flies fed with 1% H₂O₂ (B, Student's <i>t</i>-test, n = 5, ** p<0.01; C, Student's <i>t</i>-test, n = 4, * p<0.05). (D–E) DLP mRNA (D) and protein (E) levels in control (<i>elav</i>/Y) and pan-neuronally <i>dFOXO</i>-overexpressing (<i>elav</i>><i>dFOXO</i>) fly heads (D, Student's <i>t</i>-test, n = 6, *** p<0.001; E, Student's <i>t</i>-test, n = 5, * p<0.05). (F) Luciferase assays showed activation of <i>DLP</i> promoters in S2 cells after cotransfection with dFOXO-A3. (Open circle) Empty vector. (Open triangle) 0.5-kb fragment of <i>DLP</i> promoter. (Filled circle) 1.3-kb fragment of <i>DLP</i> promoter. (Filled triangle) 1.3-kb fragment of <i>DLP</i> promoter with mutation in the putative FRE site: pGL3–1.3 kb (mut). Bold characters in the putative FRE site represent the mutated nucleotides. (Student's <i>t</i>-test, n = 3, * p<0.05; ** p<0.01; *** p<0.001). (G) The levels of phospho-Akt in the head of WT and <i>DJ-1β^ex54</i> flies fed with 1% H₂O₂ (Student's <i>t</i>-test, n = 3, * p<0.05). dAkt was used as an internal control. (H) <i>d4E-BP</i>, a target of dFOXO, and <i>dFOXO</i> mRNA levels in the head of WT and <i>DJ-1β^ex54</i> flies fed with 1% H₂O₂ (Student's <i>t</i>-test: <i>d4E-BP</i>, n = 7, *** p<0.001; <i>dFOXO</i>, n = 7). (I) DLP protein levels in the control (<i>elav</i>/Y) and pan-neuronally <i>PTEN</i>-overexpressing (<i>elav</i>><i>PTEN</i>) fly heads (Student's <i>t</i>-test, n = 4, * p<0.05). (J) Genetic interactions of <i>dFOXO</i> with <i>DJ-1β</i> in the developing eye. The upper pictures are scanning electron micrographs of the fly eyes. The lower pictures are acridine orange-stained images of the eye imaginal discs. The genotypes of the samples were <i>tub</i>-<i>GAL4</i> (<i>tub</i>-<i>GAL4</i>/+), <i>tub</i>-<i>GAL4</i>, <i>DJ-1β^ex54</i> (<i>tub</i>-<i>GAL4</i>, <i>DJ-1β^ex54</i>/<i>DJ-1β^ex54</i>), <i>tub</i>><i>cncCi</i>, <i>DJ-1β^ex54</i> (<i>UAS</i>-<i>cncC</i>-<i>RNAi</i>/+; <i>tub</i>-<i>GAL4</i>, <i>DJ-1β^ex54</i>/<i>DJ-1β^ex54</i>), <i>dFOXO²¹</i>, <i>DJ-1β^ex54</i> (<i>dFOXO²¹</i>, <i>DJ-1β^ex54</i>/<i>DJ-1β^ex54</i>), <i>elav</i>/Y (<i>elav-GAL4</i>/Y), <i>elav</i>><i>dFOXO</i> (<i>elav-GAL4</i>/Y; <i>UAS-dFOXO</i>/+), <i>elav</i>><i>PTEN</i> (<i>elav-GAL4</i>/Y; <i>UAS-PTEN</i>/+), <i>ey-GAL4</i> (<i>ey-GAL4</i>/+), <i>ey</i>><i>dFOXO</i> (<i>UAS-dFOXO</i>/+; <i>ey-GAL4</i>/+), and <i>ey</i>><i>dFOXO</i>+<i>DJ-1β</i> (<i>UAS-dFOXO</i>/<i>UAS-HA-DJ-1β</i>; <i>ey-GAL4</i>/+). pAkt, phospho-Akt; <i>ey</i>, <i>eyeless</i>. All data are expressed as means ± s.e. values. Actin was used as an internal control.</p