Reporter assays were quantified using a Dual Luciferase reporter assay. SEPP1 promoter constructs cloned into pGL4.21 or a mouse mammary tumor virus promoter reporter construct (pLTRluc) were co-transfected with the pRL-RSV plasmid that serves as an internal control for transfection efficiency. Cells were seeded into 6-well plates at a concentration of 5 ×105 cells/well. Each well was cotransfected with approximately 1 μg of firefly reporter plasmid along with 50 ng of the pRL-RSV vector. Twenty four hours after transfection, medium was replaced. Cells transfected with SEPP1 promoter constructs were treated with either 10 μM of the ecdysone analong ponasterone A, 10 nM dexamethasone, or a combination of both for an additional 24 hr. Vehicle treatment with EtOH and/or DMSO served as negative controls. Cells transfected with pLTRluc were treated with either DMSO or 10 nM dexamethasone for 24 hr. Following treatments, cells were collected in 200 μl of Passive Lysis Buffer and stored at −80°C at least overnight to allow for cell membrane disruption. Cell lysates were diluted in Passive Lysis Buffer and each sample was quantified in triplicate on
