PMC

Effect of dSesn on progressive muscle degeneration. Thoracic skeletal muscles of indicated 20-day-old male flies treated without or with indicated drugs were analyzed by transmission electron microscopy (TEM). Left panels: sarcomeres; right panels: mitochondria. Mitochondrial microstructure is shown in the insets (0.15 μm width). Scale bars, 0.2 μm.

Effect of dSesn on cardiac function. (A, B, E to G) Representative M mode records of indicated 2-week-old flies fed without or with indicated drugs, showing movement of heart tube walls (y-axis) over time (x-axis). Diastolic (orange) and systolic (blue) diameters are indicated. 1 second is indicated as a bar. (C and D) Quantification of cardiac function parameters. P values were calculated using one-way ANOVA. Error bars indicate S.E.M.; n>10. (H and I) Actin fibers in WT and dSesn-null hearts were visualized by phalloidin staining (red).

Effect of dSesn on lipid homeostasis. (A) Lipid accumulation in fat bodies examined by Nile Red staining (red). (B) Total triglycerides were measured in five 10-day-old adult males of the indicated genotypes subjected to the indicated treatments (met., metformin; rapa., rapamycin). P values were calculated by one-way ANOVA. Error bars=S.D.; n≥3. (C and D) Protein lysates from fat bodies were analyzed by immunoblotting with indicated antibodies. Relative band intensities were quantified and are shown as a bar graph. Error bars=S.D.; n=3. (E) Expression of indicated mRNAs in adult flies was examined by quantitative RT-PCR. Fifty 3-day-old adult males of each genotype were used to prepare total RNA. Error bars=S.D.; n=3.

Chronic TOR activation results in accumulation of ROS and dSesn. Larval imaginal discs of indicated strains were stained as indicated. (A) ROS accumulation (red) in response to InR^CA or Rheb overexpressed in dorsal (upwards) wing discs was revealed by DHE staining. (B) InR^CA-induced ROS (red) accumulation in eye discs was reduced by PI3K^DN or TOR^DN but not by S6K^DN or 4E-BP^CA. (C) DHE staining (red) in TSC1-negative wing disc clones marked by absence of GFP. (D and E) Inhibition of InR^CA-induced accumulation of dSesn (green) in eye and wing discs by expression of catalase or peroxiredoxin (Prx). D-V wing boundary and differentiated eye area were visualized by Wg (red) and Elav (red) staining, respectively. (F) dSesn accumulation (red) in TSC1-negative wing disc clones was suppressed by vitamin E feeding. Absence of GFP (green) indicates loss of TSC1. (G) Diagram depicting TOR-stimulated production of ROS and expression of dSesn.

Antagonism of TOR-stimulated growth by dSesn. (A to F) Light (left) and scanning electron (right) micrographs of eyes expressing the indicated genetic elements driven by gmr-GAL4. Scale bar, 20 μm. (G) Quantification of eye and ommatidia sizes measured from frontal and lateral views, respectively. P values were calculated by one-way ANOVA. Error bars=S.D.; n=3 and 5, respectively. (H) Suppression of TOR signaling by dSesn. Adult heads with eye-specific expression of indicated genetic elements driven by gmr-GAL4 were subjected to immunoblot analyses with indicated antibodies. Relative band intensities were quantified and are presented as bar graphs. Error bars=S.D. n=3. (I to N) Suppression of InR-induced growth by dSesn. Anterior views of wing blades with apterous-GAL4-driven expression of indicated genetic elements. Dorsal sides point upwards. (O) Schematic diagram summarizing genetic interactions between dSesn and TOR signaling components.

Phenotypes caused by silencing of dATG1. (A and B) Representative M mode records of control and dATG1^RNAi-expressing hearts from 2-week-old adult flies, showing the movement of heart tube walls (y-axis) over time (x-axis). Diastolic (orange) and systolic (blue) diameters are indicated. 1 second is indicated as a bar. (C) Quantification of cardiac function parameters. P values were calculated using one-way ANOVA. Error bars indicate S.E.M.; n≥9. (D) dATG1^RNAi-expressing thoracic skeletal muscle was analyzed by TEM. Left panel: sarcomeres; right panel: mitochondria. Mitochondrial microstructure is shown in the insets (0.15 μm width). Scale bars, 0.2 μm. (E) ROS accumulation (red) in response to dATG1^RNAi expressed in dorsal (upwards) wing discs revealed by DHE staining.

Increased abundance of dSesn upon TOR activation. Larval wing discs of indicated strains were stained to visualize indicated proteins or mRNA. The dorsal side points upwards. Dorsoventral boundary (D/V in A) was visualized by staining with an antibody to the wingless (Wg) protein (red). (A to C) Expression of dSesn protein (green) in the absence (A) or presence of InR^CA in WT (B) and dSesn-null (C) strains. (D and E) Accumulation of dSesn mRNA (green) in response to InR^CA detected by in situ hybridization. (F) The signaling network controlling TOR activity and expression of dSesn. (G to K) Accumulation of dSesn (green) in response to Rheb (G) but not S6K^CA (J) or loss of 4E-BP (K). Thor¹ is a Drosophila 4E-BP loss-of-function mutant. (H and I) Accumulation of dSesn after somatic loss of PTEN (H) or TSC1 (I). Absence of GFP (green) indicates loss of PTEN or TSC1 resulting in dSesn (red) accumulation.