PMC

Synphilin-1 increased TAG and glycogens in adult flies. The homogenates from adult non-transgenic and ddc-GAL4;UAS-SP1 transgenic flies at 10 days of age were assayed for TAG (a, b) or glycogen (c). Triacylglycerol and glycogen levels were normalized to protein levels, and are presented relative to the level in non-transgenic control flies (pUASTattb-synphilin-1). Error bars represent s.e.m., independent samples of 40 animals each group; *P < 0.05 by analyses of variance vs non-transgenic control flies.

Synphilin-1 transgenic flies were resistance to food deprivation. Cohort of 60 flies from each group at 10 days of age were subjected to starvation resistance assays. Flies were kept at 25 °C, and dead flies were counted and removed from the vials daily. Each experiment was performed in triplicate. (a) female; (b) male. Survival data were analyzed by Kaplan–Meier log-rank survival analysis. *P < 0.05, statistically significant differences between non-transgenic and SP1 transgenic flies.

Synphilin-1 regulates lipin gene expression. Cohort of 60 flies from each group at 10 days of age were food deprived for 24h (male) or 48 h (female). The total RNA were extracted and subjected to real-time RT-PCR to detect the mRNA level of DmLpinA and DmLpinK. (a) Flies at normal fed condition. *P < 0.05 by ANOVA, statistically significant differences between non-transgenic and SP1 transgenic flies. (b, c) Flies at starvation conditions. (b) Female; (c) male. *P < 0.05 by ANOVA, statistically significant differences between normal fed and starvation group. ^#P < 0.05 by ANOVA, statistically significant differences between non-transgenic and SP1 transgenic flies.

Synphilin-1 increased food intake in adult flies. (a, b) CAFÉ assay was used to monitor fly food intake at 5 days of age for 1–5 days. The food intake of each fly was recorded daily. Data are means ± s.e.m. (a) Daily food intake; (b) 48 h cumulative food intake. Significant differences between SP1 transgenic flies and non-transgenic control mice as indicated, *P < 0.05 by analyses of variance. (c, d) Cohorts of 60 flies from each group at 10 days of age were subjected to climbing assay (c) and the actometer assay (d) to measure locomotor activity. Shown are representative data from three separate experiments.

Synphilin-1 increases adult fly body weight. (a) Schematic of site-specific integration between the attB and attP sequences to generate UAS-SP1 fly line. Human synphilin-1 cDNA with a myc tag was cloned into vector pUASTattB containing attB site. Site-specific integration of the resulting construct into attP40 flies at attP lading site was carried out by coinjection with phiC31-integrase RNA. (b) Western blot analysis showing human synphilin-1 protein expression in various fly tissues under indicated tissue-specific GAL4 promoter drivers using anti-synphilin-1 antibodies. (c, d) Human synphilin-1 was expressed by combination of the pUASTattB-synphilin-1(UAS-SP1) with Fru-GAL4, ddc-GAL4, TPH-GAL4 or Elav-GAL4 driver in Fru-GAL4 neurons, dopaminergic and seronergic neurons, and all neurons, respectively. The body weight of flies was measured at 10 days of age. There were 70 flies in each group. (c) female; (d) male. Data are means ± s.e.m. Significant differences between SP1 transgenic flies and non-transgenic control mice as indicated, *P < 0.05 by analyses of variance.

Synphilin-1 increased fat storage in the third larvae stage. (a, b) SP1 increased fat storage in the third instar larvae using a buoyancy-based density assay. The non-transgenic and SP1 larvae were immersed in the 9% sucrose solution in plastic cuvettes and photographed after reaching equilibrium. (a) Images of floating assays. (b) For the indicated genotypes, mean floatation scores (% floating larvae) were calculated from three independent replicates, for each using ~ 20 larvae submerged in sucrose. Error bars represent s.e.m. *P < 0.05 by analyses of variance (ANOVA). Significant differences between SP1 transgenic flies and non-transgenic controls (UAS-SP1 or ddc-GAL4) as indicated. (c–f) Synphilin-1 was expressed using various GAL4 drivers as indicated. (c) Representative images of fat body cells from wandering third instar larvae in each experimental group stained with phalloidin (red) and DAPI (blue) for nuclei. (d) Quantitation of fat body cell size was carried out with Image-J software. Significant differences between SP1 and non-transgenic control larvae as indicated, *P < 0.05 by ANOVA. (e) Representative fluorescent microscopy images of lipid droplets in fat body cells from third instar larvae in each experimental group stained with nile red. Top: excitation: 515–560 nm, emission: 590 nm; bottom: excitation: 450–500 nm, emission: 528 nm. (f) The body size of the third instar larvae was measured. Data are means ± s.e.m. There were 10 larvae in each group. Significant differences between SP1 and non-transgenic control larvae as indicated, *P < 0.05 by ANOVA.