Figure 1.

Regulation of miR-8 and USH by ecdysone signaling in vivo and in vitro. (A, top) Temporal expression pattern of miR-8 during larval development. (Bottom) Temporal expression patterns of E74 and BR-C during larval development. Harvesting times (AEL) are denoted above the blot. (B) Temporal expressions of miR-8 from two independent experiments are individually plotted as blue and red lines. (C) Temporal expression of ush transcripts from two independent experiments are individually plotted as blue and red lines. (D) Feeding with 20-hydroxyecdysone (20E) decreases the level of miR-8 in the whole larvae. Newly hatched larvae were fed 20E-containing (0.25 mg/mL) glucose–yeast paste until 3 d AEL, after which RNA was prepared. (E) Inhibition of EcR activity by expressing EcRDN in larval fat bodies increases miR-8 levels sustainably during the third instar larval period. (F) EcRDN expression in larval fat bodies sustainably decreases USH transcripts during the third instar larval period. Two biological replicates (six to nine animals from each genotype per replicate) were measured. (G) Fat cell clones overexpressing EcRDN by FLP-out GAL4 (marked by the absence of CD2; white arrows) show decreased levels of USH, as analyzed by immunostaining with USH antibody. (H) Treatment with 10 μM 20E in S2 cells progressively decreases both mature and pri-miR-8 levels in a similar pattern. pri-miR-8 levels were normalized against mitochondrial large ribosomal RNA1 (mtl rRNA1). This experiment was repeated with similar results. (I) Knockdown of EcR, E74, or BR-C impedes the repressive effect of 20E on pri-miR-8 expression in S2 cells (n = 3). (J) Promoter activity of pri-miR-8 is significantly down-regulated by 20E treatment. The inset shows a magnified graph of the pGL3-null reporter. Fivefold more pGL3-null plasmids were transfected than the other reporter plasmids (n = 3). (***) P < 0.001; (**) P < 0.02; (*) P < 0.05 compared with respective controls. (n.s.) Not significant (P > 0.3). Error bars denote SEM.