FIGURE 3.

Effects of FXR silencing on aromatase expression in R2C cells. A, FXR protein in R2C cells that were not transfected (−) or transfected with siRNA targeted rat FXR mRNA sequence as reported under “Experimental Procedures” for 24, 48, and 72 h. GAPDH was used as loading control. The histograms represent the mean ± S.D. (error bars) of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary units and expressed as percentages of the control, which was assumed to be 100%. *, p < 0.01 compared with not transfected cells. B–D, R2C cells were transfected with control siRNA or FXR siRNA for 24 h, and then treated with vehicle (−), 50 μm CDCA, or 3 μm GW4064 for 24 h. B, total RNA was extracted and reverse transcription-PCR analysis was performed to evaluate the expression of aromatase. L19 was used as loading control. NC, negative control, RNA sample without the addition of reverse transcriptase. C, total proteins were extracted and Western blotting analysis was performed. GAPDH was used as loading control. The histograms represent the mean ± S.D. of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary units and expressed as percentages of the control, which was assumed to be 100%. *, p < 0.01 compared with vehicle. D, aromatase activity was performed as described under “Experimental Procedures.” The results obtained were expressed as picomole of [³H]H₂O/h of release and were normalized for milligrams of protein (pmol/mg of proteins/h). The values represent the mean ± S.D. of three different experiments each performed with triplicate samples. *, p < 0.01 compared with vehicle.