Fig. 5.

The putative NF-κB enhancer of CRH regulates transcription of a heterologous promoter. A, Schematic presentation of luciferase reporter constructs. pGL4.32 (Promega) harbors five copies of the NF-κB enhancer and a minimal promoter (TATA), which regulate expression of firefly luciferase gene (FL). pGL4.32-CRH1X and -CRH4X contain one and four copies of 5′-GGGAAATCTC-3′, respectively. pGL4.32-CRH1XMut that contains one copy of mutant version (mutant residues underlined) was also created. pCMV-RL (renilla luciferase) was used as the internal control. B, Human cytotrophoblasts were cotransfected with pCMV-RL (200 ng) and the constructs (1 μg) as indicated at the bottom. The bars represent the average of fold change of FL with normalization to renilla luciferase (RL), and error bars represent the sd from three experiments. Fold FL activity by pGL-4.32CRH1XMut was defined as 1. *, P < 0.001, **, P < 0.01 (vs. FL activity of control cells transfected with pGL4.32-CRH1XMut), as determined by one-way ANOVA with Dunnett's test. C, Cytotrophoblasts were cotransfected with pGL4.32-CRH1X (1 μg) and pCMV-RL (200 ng) for 24 h and then treated with vehicle (−) or with DEX or progesterone (Prog) for an additional 24 h. FL activity was determined with normalization to RL activity and presented in bars and errors as described in B. Fold FL activity by pGL-4.32CRH1X in the presence of vehicle but absence of steroids was defined as 1. *, P < 0.001, **, P < 0.01 (vs. FL activity of control cells transfected with pGL4.32-CRH1X in the presence of vehicle), as determined by one-way ANOVA with Dunnett's test.