Fig. 1

Inhibition of ERβ activity by oleuropein a Natural product screening for ERβ-selective inhibitors. HeLa cells were transiently transfected with ERβ expression vector and ERE-luciferase reporter. The ratio of luciferase activity upon E2 (10 nM) administration plus natural product (10 nM) or E2 (10 nM) alone was determined. Oleuropein (10 nM) significantly inhibited ERβ activity compared with the vehicle (arrowhead). b, c Dose-dependent effect of oleuropein on ERβ and ERα activity. HeLa cells were transiently transfected with ERβ (b) or ERα (c) expression vector plus ERE-luciferase reporter. The luciferase activity was determined upon treatment with E2 (10 nM) plus various concentrations of oleuropein. d-g Immunofluorescence staining of ERβ in IHEECS:ERB cells upon treatment with the vehicle (d), 10 nM E2 (e), 10 nM oleuropein (f), and 10 nM E2 plus 10 nM oleuropein (g) for 24 h. h Screening of natural products for ERβ protein degradation. HeLa cells were transiently transfected with an expression plasmid for the luciferase-ERβ fusion protein, and the ratio of luciferase activity after treatment with natural products (10 nM) or the vehicle was determined at 24 h posttreatment. The arrowhead indicates oleuropein. i Effect of oleuropein on ERβ protein stability. Primary human endometriotic stromal cells were treated with different doses of oleuropein, and ERβ protein levels were determined by Western blot analysis at 24 h after treatment. The tubulin level determined the protein loading amount el. j Expression profile of phospho kinases involved in MAPK pathways in ectopic HESCs treated with vehicle or 10 and 100 nM OLE for 24 h. k Quantification of the ratio of phospho-MEK to the loading control in Panel j. OLE, oleuropein. *, p < 0.05; **, P < 0.01; ***, p < 0.001; NS, nonspecific. Scale bar is 200 μm