Effect of recombinant pigment epithelium-derived factor on the estrogen receptor alpha signaling pathway. (a) Western blot analysis of pigment epithelium-derived factor (PEDF), estrogen receptor alpha (ERα), phospho-ER, phospho-Akt, mitogen-activated protein kinase (MAPK), phospho-MAPK, rearranged during transfection (RET), pRET, p70S6K, and mammalian target of rapamycin (mTOR) protein expression in MCF-7, MCF-7:5C, and 5C-PEDF cells. (b) Effect of recombinant PEDF on ERα, pERα, RET, and pRET (Y1062) protein expression in MCF-7:5C cells. Cells were treated with 100 nM recombinant PEDF (rPEDF) protein for 24 hours and cell lysates were analyzed by western blot. β-actin was used as a loading control. (c) Effect of rPEDF on estrogen response element (ERE) luciferase activity in MCF-7 and MCF-7:5C cells. MCF-7 and MCF-7:5C cells were grown in estrogen-free RPMI media and then co-transfected with a 5× ERE luciferase plasmid and a renilla reporter plasmid for 24 hours. Following transfection, cells were treated with 1 nM 17β-estradiol (E2), 100 nM rPEDF, or E2 + rPEDF for 24 hours and luciferase activity was measured. Values presented as relative luciferase activity after normalization to Renilla luciferase activity. Data expressed as mean ± standard deviation of the results obtained from triplicate experiments. Basal ERE activity was statistically significantly higher in MCF-7:5C cells compared with MCF-7 cells. *P < 0.01.