Fig. 6.

The effects of RelB/NF-κB2 gene silencing on transcriptional and translational levels of CRH in primary cytotrophoblasts. A, Human term cytotrophoblasts were transfected with RelB or NF-κB2 siRNA or nontargeting scramble siRNA (SS) for 24 h. The cells were then treated with vehicle (−) or with DEX 1 μm or progesterone (Prog) 10 μm. After another 24 h, the cells were lysed and probed with antibody to human CRH, RelB (left panel), or NF-κB2 (right panel). β-Actin served as a loading control. B, Total RNA were extracted from A, and quantitative RT-PCR was performed with normalization to GAPDH mRNA. The bars represent average of fold change in mRNA levels, and error bars represent sd from three experiments. The mRNA levels of RelB or NF-κB2 in cells treated nontargeting scramble siRNA in the absence of steroids were defined as 100%. *, P < 0.001, **, P < 0.01 (vs. that of control cells transfected with SS and treated with vehicle), as determined by one-way ANOVA with Dunnett's test. C, The cells pretransfected with siRNA indicated in A were cotransfected with pGL4.32-CRH1X (1 μg) and pCMV-RL (200 ng) and with vehicle or DEX or progesterone (Prog) for an additional 24 h. FL activity was determined with normalization to renilla luciferase (RL) activity. The bars represent average of percentage change in FL activity, and error bars represent sd from three experiments. FL activity in cells treated nontargeting scramble siRNA in the presence of vehicle but absence of steroids was defined as 100%. *, P < 0.001, **, P < 0.01 (vs. that of control cells transfected with SS and treated with vehicle), as determined by one-way ANOVA with Dunnett's test.