Pigment epithelium-derived factor expression in human breast cancer cell lines. (a) Pigment epithelium-derived factor (PEDF) and estrogen receptor alpha (ERα) protein levels were detected by western blot analysis in several human breast cancer cell lines, including hormone-dependent T47D, MCF-7, and ZR-75-1 cells, tamoxifen-resistant BT474 cells, tamoxifen-resistant and aromatase inhibitor-resistant MCF-7:5C and MCF-7:2A cells, and ER-negative MDA-MB-231 cells. (b) PEDF mRNA expression level in the different breast cancer cell lines was determined by quantitative real-time PCR analysis with PUM1 used as a normalization control. (c) Conditioned media were analyzed by western blot for the presence of PEDF protein in the indicated breast cancer cell lines. Staining of proteins by Coomassie blue included as a loading control. (d) Hormonal regulation of PEDF and ERα protein in MCF-7 and T47D breast cancer cells. Cells were incubated in phenol red-free RPMI media supplemented with 10% charcoal-stripped FBS for 72 hours and, subsequently, were treated with vehicle, 1 nM 17β-estradiol (E2), or 1 nM E2 + 1 μM 4-hydroxytamoxifen (4OHT) for 24 hours, and the PEDF and ERα protein level was determined by western blot analysis. β-actin was used as a normalization control. All experiments were performed in triplicate independently.
Jan et al. Breast Cancer Research 2012 14:R146 doi:10.1186/bcr3356