In vitro activity: PHTPP is an antagonist of the ERβ (Estrogen Receptor). It is selective for the with 36-fold selective for ERβ over ERα. PHTPP reduces FSH-mediated cAMP production by 80% while has no effect on basal cAMP. PHTPP (at concentration of 10-6 M) inhibits E2-stimulated ERβ activity, but does not suppress E2-stimulated ERα activity. At a high dose, PHTPP slightly increases class 1 Igf1 mRNA expression, and facilitates the DPN-induced increase in class 1 Igf1 mRNA expression.

Kinase Assay:

Cell Assay: Cells are seeded in the plate at a density of 3 to 4×105 in 1 mL of medium per well in a 24-well plate for cell viability and cAMP analysis. Cells are cultured in Dulbecco’s Modified Eagle Medium with PHTPP (1 μM), or ethanol (0.1%) as the vehicle. The incubator is set to an atmosphere of 5% CO2 in air at 37°C, and cultures are allowed to acclimate for at least 24 h. The culture medium is then aspirated and replaced with serum-free DMEM/F12 containing 0.1 μM androstenedione. The cells are collected for intracellular cAMP and to test cell viability.