White bar indicates lights-on and dark bar and gray highlight indicate lights-off. (A) Bar diagram indicating general activity over a 48 hour period of adult flies maintained on a 12:12 hour light:dark cycle. The DAMS system was used to monitor locomotion and sleep/wake behaviour over a 48 hour period. (C-E: n = 30–60 males per strain * P < 0.05, ** P<0.01, *** P < 0.005 compared with controls, one-way ANOVA with Tukey’s post hoc test for multiple comparisons) In all graphs error bars = SEM.Įts96B regulates activity and sleep/wake behaviour. Only Ets96B RNAi1 was used for this assay. (E) The DAMS system was used to monitor locomotion prior to and after sound stimulation (65–70 dB). (D) The DAMS system was used to monitor locomotion prior to and after light stimulation. (C) The DAMS system was used to monitor locomotion for the first four hours flies were placed in the system. ![]() (A, B: n = 50 males per strain ** P<0.01 compared with controls, one-way ANOVA with Tukey’s post hoc test for multiple comparisons). (A) Ets96B knocked down throughout development using UAS- Ets96B RNAi1 or UAS- Ets96B RNAi2 crossed to the pan-neuronal driver elav-GAL4 (B) Ets96B knocked down only in adults using UAS- Ets96B RNAi1 crossed to the pan-neuronal driver and temperature sensitive allele of the GAL4 inhibitor GAL80 elav-GAL4, tub-Gal80 ts. (A, B) Startle-response test demonstrating Ets96B knockdown males have a hyperactive startle-response. Error bars = SEM.Įts96B knockdown during development induces a heightened startle-response. (D, E: n = 30 males per treatment, assay was repeated at least 10 times for each genotype, one-way ANOVA with Tukey’s post hoc test for multiple comparisons). The (D) elav-Gal4 driver was used to express Ets96B RNAi ( Ets96B RNAi1 or Ets96B RNAi2 ) throughout development (UAS- Ets96B RNAi1 and UAS- Ets96B RNAi2) and (E) the elav-GAL4, tub-GAL80 ts driver was used to knockdown Ets96B ( Ets96B RNAi1 or Ets96B RNAi2 ) in adult males. Flies fed ad lib were set as 100%, represented by 1 on the graphs (B, C: n = 10 replicates, one-way ANOVA with Tukey’s post hoc test for multiple comparisons) (D, E) Triglyceride levels were determined in male flies at 0, 12 and 24 hours of starvation. (C) To examine how nutritional state affects the expression levels of Ets96B, RNA was extracted under different nutritional states. ![]() (n = 30–60 flies per genotype, one-way ANOVA with Tukey’s post hoc test for multiple comparisons) (B) To examine how starvation affects the expression level of Ets96B, RNA was extracted from normal fed flies, as well as after 24 and 48 h of starvation. 5–7 day old control and Ets96B knockdown males (GAL4 driver crossed to either Ets96B RNAi1 or Ets96B RNAi2) were placed in a vial containing 1% agarose and maintained at 25☌. (A) We tested the effect of Ets96B-knockdown in flies using the starvation survival assay. Drosophila homologues that were up-regulated when Ets96B was knocked down are in red, and those that were up-regulated more than 2.5-fold are underlined. These include molecular chaperones that function to prevent aggregation (Bip, HSP90B1, DNAJC3, HYOU1 and CALR), protein disulfide isomerases that catalyze disulfide bond formation, isomerization and reduction (PDIA3, PDIA3 and P4HB), and proteins involved in de- or re-glycosylation of improperly folded glycoproteins (PRKCSH and UGGT1). ![]() (E) Diagram depicting molecules involved in endoplasmic reticulum protein folding. Genes in red were recovered in the Ets96B SOLiD sequencing. (D) Data showing human homologues of Drosophila genes recovered in Biogrid and String analysis. ![]() (C) Biogrid and String were used to find all genes that interact with the molecular chaperone genes recovered in the Ets96B knockdown males. (n = 25 males per treatment *** P<0.005 compared with elav-GAL4 controls, one-way ANOVA with Tukey’s post hoc test for multiple comparisons). This assay was repeated at least 7 times. (B) Relative expression levels of CaBP1, ERp60 and Crc in 5–7 day old control males or males where Ets96B was knocked down in the entire nervous system throughout development. Only genes regulated more than ±log2 2.5-fold change are included. Ets96B RNAi versus controls ( w, elav-GAL4> w 1118 and w 1118> Ets96B RNAi) 5–7 day old male heads.
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