Journal: Nature communications
Article Title: An ecdysone-responsive nuclear receptor regulates circadian rhythms in Drosophila
doi: 10.1038/ncomms6697
Figure Lengend Snippet: (A) Mammalian HEK-293T cells were transfected with Clk -luc (50ng) and renilla -luc (10ng) reporter (internal control) and with increasing doses of CMV- Pdp1ε (10 and 50 ng) or CMV- E75 (50, 100, and 250 ng) and in some cases with CMV- per (10, 50, 100 ng). The (+), (++) and (+++).denote 10, 50, and 100 ng of DNA respectively, except for the empty pCDNA3.1 vector. Additionally, CMV- gfp (10, 50, 100 ng) was used a control for per . The firefly luciferase activity was normalized to renilla -luc activity, and the fold induction (Y axis) was based upon comparisons with 10 ng of CMV expression vector alone transfections. Data represent an average of four experiments each performed in duplicate. Error bars depict standard error of the mean. C3m-TK luc (a mutant promoter, which cannot be activated by PDP1), was used as an additional control. PDP1 ε and E75 significantly activated and repressed Clk -luc respectively, compared to empty vector pCDNA3.1 controls. PER significantly de-repressed the E75 mediated repression of Clk promoter repression. (B) An artificial promoter, C3- TK luc, was used instead of the native Clk -luc promoter to determine if E75 acts as a co-repressor with VRI. C3- TK luc contains 3 tandem consensus binding sites for PDP1/VRI but none for E75. Other details, including the dosage used, are same to panel (A) . PDP1 activates this promoter in a dose dependent manner, and this is repressed by VRI. The highest dose of E75 enhances repression by VRI. (*p < 0.05. Two-way ANOVA, Tukey’s test post-hoc comparison).
Article Snippet: Mammalian HEK293T cells (~2 × 10 6 ) were used were transfected with the following plasmids- pcDNA3- per -HA, pcDNA3.1 E75 , pcDNA3- cry , pcDNA3.1 Gaba-t using Lipofectamine (Life Technologies) reagent and cells were harvested and lysed after 48hrs.
Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Mutagenesis, Binding Assay
