Fig. 10.

Knockdown of Creb and c-Fos reduced luciferase activity of Aanat (−484+120)luc reporter-transfected retinal cells. Cells were transfected with 50 ng of Creb and c-Fos siRNAs. For each well of cells, 1.0 μg of the Aanat (−484+120)luc construct was co-transfected with 0.1 μg of pRL-TK vector that provides constitutive expression of Renilla luciferase serving as an internal control. Cells with vectors and siRNA’s were incubated for 24 hours, and were stimulated by forskolin at a final concentration of 10 μM for 6 hours before harvesting. Luciferase activities were determined by dual luciferase reporter assay system. Levels of firefly luciferase expression were normalized to Renilla luciferase activity of co-transfected pRL-TK plasmid. The luciferase activity of the Aanat promoter construct is expressed as fold change over pGL3-basic vector. Values are presented as mean ± SEM, n=3–4. Two-way ANOVA indicated a significant effects of treatment (vehicle vs forskolin; p<0.001), siRNA (p<0.001), and a significant interaction of treatment and siRNA (p<0.001);, **p<0.001 vs. control; #p<0.05 vs. Creb siRNA or c-Fos siRNA; ##p<0.05 vs. Creb siRNA + forskolin or c-Fos siRNA + forskolin. For further details see Materials and Methods.