FIGURE 4. MPA increases ERα degradation in ECC1 and USPC1 cell lines.

ECC1 and USPC1 cells were plated in 6-well plates, and after 24 hr they were treated with FTS, MPA, FTS + MPA (concentrations as in Fig. 2), or 0.1% DMSO (control) (four wells per treatment). After 5 days, one well from each treatment was lysed (time zero) and the other three wells were treated with CHX (50 μg/ml). At 6, 24, or 32 hr after CHX addition the cells were lysed and subjected to western blot analysis with anti-ERα antibody. β-tubulin was used as loading control. Results of a typical experiment are presented, showing ERα and β-tubulin expression levels (a) in ECC1 cells and (b) in USPC1 cells. The blot was loaded with samples of control, FTS-treated, MPA-treated, and FTS + MPA-treated cells taken at time zero and at 6, 24 and 32 hr after addition of CHX. (c) Statistical analysis of ERα after treatment with CHX for 32 hr, presented as percentages of ERα at time zero, for each of the treatments of ECC1 cells and (d) of USPC1 cells. ERα stability in both cell lines was significantly reduced after treatment with MPA and with FTS + MPA, suggesting that ERα degradation is increased by MPA but not by FTS. Results are shown as means ± SEM (n = 4). * and ** are compared with ERα levels, 32 hr after CHX addition, expressed as a percentage of the control at time zero in each cell line. *p < 0.05, ** p < 0.01. CHX, cycloheximide; Con, control; ERα, estrogen receptor alpha; FTS, S-farnesylthiosalicylic acid; MPA, medroxyprogesterone acetate.