Transient Transfection of MDA-MB-231 Cells with pGRE-SEAP Reporter

MDA-MB-231 cells were transiently transfected with a pGRE-SEAP reporter construct (Clontech, Mountain View, CA, USA) as previously described57 (link). Briefly, cells were transfected for 6 hours with a pGRE-SEAP reporter construct (150 ng/well) that was complexed with Lipofectamine 2000 reagent (1 μL/well) in 100 μL/well of OptiMEM for 20 min at room temperature prior to addition to cells. The day after transfection, cells were treated with RU486 (1 μM) for 30 min prior to glucocorticoid stimulation. After 24 h, supernatants were collected and the amount of secreted human placental alkaline phosphatase (SEAP) was determined using a chemiluminescence kit (Roche Applied Science, NSW, Australia).