Figure 7.

Effects of FXR ligand on SKBR3 breast cancer cells. (a) MTT proliferation assay of SKBR3 cells treated with vehicle (−) or increasing doses of CDCA (12,5–25–50–100 µM) for 7 days. Results are expressed as fold change ± SD relative to vehicle treated cells, and are representative of three different experiments each performed in triplicate. (b) Soft Agar growth assay in SKBR3 cells plated in 0.35% agarose and treated with vehicle (−) or CDCA 50 µM. After 14 days of growth colonies > 50 µm diameter were counted. (c) SKBR3 cells were treated as indicated with vehicle (−) or CDCA 50µM before lysis. Equal amounts of total cellular extract were analyzed for HER2 levels by Western blotting. β-Actin was used as loading control. Numbers on top of the blots represent the average fold change relative to control normalized for β-Actin. (d) mRNA HER2 content, evaluated by real time RT-PCR, after treatment with vehicle (−) or CDCA 50µM as indicated. Each sample was normalized to its GAPDH mRNA content. (e) SKBR3 cells were transiently transfected with pNeuLite construct. After transfection cells were treated in the presence of vehicle (−) or CDCA 50 µM for 24h and the promoter activity was evaluated. The values represent the means ± SD of three different experiments each performed in triplicate. * p<0.05 compared to vehicle. (f) MTT growth assay in MCF-7TR1 and MCF-7/HER2-18 cells treated with vehicle (−), CDCA 50µM and GW4064 3µM in the presence or not of Tam 1µM for 4 days. Results are expressed as fold change ± SD relative to vehicle treated cells, and are representative of three different experiments each performed in triplicate. * p<0.05 compared to Tam.