DOR moves out of the nucleus in response to the activation of autophagy. (A) The intracellular distribution of DOR is modified by starvation. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal), HBSS (starvation), 50 μM chloroquine or 10 mM 3MA. The intracellular localization of DOR was analysed by immunofluorescence and is shown in red. The nuclei are shown in blue (DAPI staining). Scale bars, 10 μm. (B) Endogenous DOR leaves the nucleus under starvation. C2C12 myoblasts were incubated for 30 min at 37°C with DMEM (basal) or HBSS (starvation). DOR is shown in red and nuclei are in blue (DAPI staining). Scale bars, 5 μm. (C,D) De-activation of autophagy returns DOR to the nucleus. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal) or HBSS (starvation). Cells were then incubated with normal DMEM for an additional hour (starvation+GM). The intracellular distribution of DOR was determined by immunofluorescence. DOR is shown in red and the nuclei are in blue. Scale bars, 5 μm. Cell lysates were also obtained and western blot assays were performed with specific antibodies. 3MA, 3-methyladenine; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes- and obesity-regulated gene; HBSS, Hank's balanced salt solution; LC3, microtubule-associated protein 1A/1B-light chain 3.

DOR stimulates autophagy. (A) DOR gain-of-function accelerates the degradation of proteins of middle–long half-life. Untreated HeLa cells and those transiently transfected with DOR were incubated in DMEM or HBSS. ^*Significant effects of starvation, P<0.01; ^τsignificant effects caused by DOR overexpression, P<0.01. (B) DOR overexpression enhances autophagosomal formation. LC3-GFP-positive vacuoles were counted in 100 transfected HeLa cells (with GFP-LC3 and TRα1, or GFP-LC3 and DOR). ^*Significant effects of starvation, P<0.001; ^τsignificant effects of DOR overexpression, P<0.001. (C,D) HeLa cells transiently transfected with empty pCDNA3 or DOR were incubated in DMEM (basal) or in HBSS (starvation). Cells were processed by transmission electron microscopy. Arrows indicate autophagosomes. Insets are autophagosomes (diameter, 407±63 nm). Scale bars, 1 μm. Autophagosomes were counted in 50 randomly chosen transfected cells. ^*Significant effects of starvation, P<0.05; ^τsignificant effects of DOR overexpression, P<0.05. DMEM; Dulbecco's modified Eagle medium; DOR, diabetes- and obesity-regulated gene; GFP, green fluorescent protein; HBSS; Hank's balanced salt solution; LC3, microtubule-associated protein 1A/1B-light chain 3.

CG11347 is required for autophagy activation in the fat body of Drosophila. (A) Decrease of mRNA levels in TubG4, UAS-CG11347-RNAi animals compared with TubG4 controls. All measurements were normalized to rp49. ^*Significant change, P<0.05. (B) Lysotracker staining of fat bodies. No Lysotracker staining is observed in feeding early third-instar larvae, whereas Lysotracker-positive granules (red) accumulate in fat body cells of wandering late third-instar controls (upper panels, TubG4/+). dDOR knockdown animals (TubG4, UAS-CG11347-RNAi T4, lower panels) show decreased staining of Lysotracker-positive granules in wandering late third-instar larvae. Nuclei are shown in blue. Scale bars, 50 μm. (C) The number of Lysotracker-positive puncta per unit area are shown for each genotype (control and RNAi), normalized to the value for control cells processed in parallel. ^*Significant change, P<0.05. (D) Transmission electron microscopy images of fat body cells from wandering late third-instar larvae. Developmental autophagy results in the accumulation of large autolysosomes (AL) in control (TubG4/+) larvae, whereas only a few autolysosomes are seen in CG11347 RNAi fat body cells of the same age. Control and RNAi fat bodies present lipid droplets (LP) with a ‘striped' appearance, which is an electron microscopy artefact. Scale bars, 5 nm. dDor, CG11347, a homologue of DOR; DOR, diabetes- and obesity-regulated gene; mRNA, messenger RNA; wt, wild type.

DOR localizes to autophagosomes when autophagy is activated. (A) Confocal images of HeLa cells transiently transfected with DOR and GFP-LC3, and incubated for 1h with DMEM, HBSS (starvation), 2 μM rapamycin or HBSS containing 50 μM chloroquine. Nuclei were labelled with DAPI. Scale bars, 10 μm. (B,C) Confocal images of HeLa cells transiently transfected with DOR or GFP-LC3 and incubated with 50 nM Lysotracker in DMEM or HBSS. Scale bars, 10 μm. (D) Confocal images of HeLa cells transiently transfected with DOR and LAMP1-GFP, or with YFP-LC3 and LAMP1-GFP. Scale bars, 10 μm. Contrast-corrected merge RGB pictures and the Z-projection of Product of the differences from the mean (PDM) images are shown in all panels. Colour scales of PDM images have different maximal values, so the different conditions are not comparable from these projections. Product of the differences from the mean (PDM) values closer to 1 show reliable colocalized pixels. DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes- and obesity-regulated gene; GFP, green fluorescent protein; HBSS, Hank's balanced salt solution; LAMP1, lysosomal-associated membrane protein 1; LC3, microtubule-associated protein 1A/1B-light chain 3.

DOR loss-of-function reduces autophagy. (A,B) C2C12 myoblasts were previously infected with lentiviruses encoding scrambled RNA, DOR siRNA1 or DOR siRNA2. Endogenous LC3 was detected by immunofluorescence. LC3-positive puncta were counted in 100 cells per group under basal conditions or starved for 20 min. Scale bars, 10 μm. ^*Significant effects of starvation, P<0.001; τsignificant effects caused by DOR knockdown, P<0.001. (C,D) Scramble or DOR siRNA1 C2C12 cells were processed by transmission electron microscopy. Arrows indicate autophagosomes. Insets are autophagosomes. Scale bars, 1 μm. Autophagosomes were counted in 50 randomly chosen cells. ^*Significant effects of DOR repression, P<0.001. (E) Scramble or DOR siRNA1 C2C12 cells were incubated in DMEM with and without 10 mM 3MA. ^*Significant effects of DOR knockdown, P<0.01; τsignificant effects caused by 3MA, P<0.01. 3MA, 3-methyladenine; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes- and obesity-regulated gene; LC3, microtubule-associated protein 1A/1B-light chain 3.