Figure 6. Har-CREB, -c-Myc and -POU regulate Har-HK promotor activity and gene expression.

(A) Har-CREB, -c-Myc and -POU expression in HzAm1 cells. Total RNA was extracted from cells and reverse transcribed to generate cDNA for the amplification of c-Myc, CREB, POU, and an acting control. (B) and (C) RNA interference directed against Har-CREB and –c-Myc reduces Har-HK promoter activity. The Har-HK promoter reporter plasmid (0.2 μg) was co-transfected with various amounts of Har-CREB or -c-Myc dsRNA (0.2 and 0.4 μg); 0.4 μg of dsGFP was used as a control. Luciferase activity was detected 48 h following transfection. (D) Har-POU activated the Har-HK promotor. HzAm1 cells were co-transfected with Har-HK promoter reporter plasmid (0.2 μg) and luciferase reporter vector (pGL3)-basic with or without 0.2 or 0.4 μg of Har-POU plasmid. Luciferase activity was detected 48 h following transfection. (E) Changes in Har-HK mRNA due to Har-CREB, -c-Myc and -POU overexpression. HzAm1 cells were transfected with plasmids encoding recombinant Har-CREB, -c-Myc, -POU, and GFP, and Har-HK mRNA was detected by qPCR using actin as an internal standard. (F) Ecdysteroid (20E) regulates Har-HK expression by controlling Har-POU protein levels. 1 μg of 20E was injected into day 20 diapausing pupae, and changes in brain Har-HK and -POU protein levels were detected at 24 h and 48 h following injection. Brain proteins (20 μg) were separated and detected with the appropriate antibodies. Con, injection of 2 μl ethanol; 20E, injection of 2 μl 20E (0.5 μg/μl, dissolved in ethanol). The western blotting bands were quantified using ImageJ software and normalized to Har-actin levels. Each point represents the mean ± S.D. of three independent replicates. The * denotes a p value < 0.05, and ** denotes a p value < 0.01 as determined by the independent t-test.