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. 2009 Dec 21;285(8):5581–5593. doi: 10.1074/jbc.M109.052670

FIGURE 8.

FIGURE 8.

CDCA reverses the effects of AD on R2C cell proliferation. A, R2C cells were transfected with control siRNA or FXR siRNA for 24 h and then transiently transfected with the XETL promoter plasmid. Cells were treated with 50 μm CDCA in the with or without 100 nm AD for 24 h. These results represent the mean ± S.D. of three different experiments. In each experiment, the activities of the transfected plasmids were assayed in triplicate transfections. *, p < 0.01 with respect to the vehicle. **, p < 0.01 CDCA + AD treated versus AD alone. B, R2C cells were treated with 100 nm AD in the presence or not of 50 μm CDCA for 24 h. Thymidine incorporation assay was performed. The results represent the mean ± S.D. of three different experiments each performed with triplicate samples. *, p < 0.01 AD treated compared with vehicle. **, p < 0.01 CDCA + AD treated versus AD alone. C, R2C cells were seeded (10,000/well) in 0.5% agarose and treated as described above. Cells were allowed to grow for 14 days and then the number of colonies >50 μm were quantified and the results graphed. The results represent the mean ± S.D. of three different experiments each performed with triplicate samples. *, p < 0.01 AD treated compared with vehicle. **, p < 0.01 CDCA + AD treated versus AD alone. D, total proteins extracted from R2C cells treated with vehicle (−), 100 nm AD, 50 μm CDCA, and AD + CDCA for 24 h were used for immunoblot analysis of cyclin D1 and cyclin E. β-Actin was used as a 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 AD treated compared with vehicle. **, p < 0.01 CDCA + AD treated versus AD alone. E, aromatase protein in R2C cells that were not transfected (−) or transfected with siRNA targeted rat aromatase mRNA sequence as described under “Experimental Procedures” for 24, 48, and 72 h. GAPDH was used as loading control. F, R2C cells were transfected with control siRNA or Arom siRNA for 48 h and then treated with 100 nm AD in the presence or not of 50 μm CDCA for 24 h. Thymidine incorporation assay was performed. The results represent the mean ± S.D. of three different experiments each performed with triplicate samples. *, p < 0.01 AD treated compared with vehicle. **, p < 0.01 CDCA + AD treated versus AD alone.