Antagonist activity at human progesterone receptor beta expressed in human U2OS cells assessed as inhibition of D(-)-norgestrel-induced protein interaction with steroid receptor co-activator peptide after overnight incubation by beta-galactosidase reporter gene assay
Antagonist activity at Gal4 DNA binding domain-tagged human progesterone receptor ligand binding domain expressed in HEK293 cells assessed as inhibition of receptor-mediated transcriptional activity by luciferase reporter gene assay
Agonist activity at Gal4 DNA binding domain-tagged human progesterone receptor ligand binding domain expressed in HEK293 cells assessed as activation of receptor-mediated transcriptional activity by luciferase reporter gene assay
Displacement of [3H]-progesterone from human progesterone receptor expressed in HEK293 cells by scintillation counting based radioligand competition binding assay
Receptor Binding Assay: Using cytosol from progesterone receptor-expressing insect cells (Hi5), competitive binding to the progesterone receptor was determined from the ability to displace 3H-progesterone as reference substance from the receptor. If a compound has an affinity corresponding to progesterone, this corresponds to a competition factor (CF) of 1. CF values greater than 1 are characterized by a lower affinity for the progesterone receptor, and CF values of less than 1 are characterized by higher affinity.
Binding Assay: Using cytosol from progesterone receptor-expressing insect cells (Hi5), competitive binding to the progesterone receptor was determined from the ability to displace 3H-progesterone as reference substance from the receptor. If a compound has an affinity corresponding to progesterone, this corresponds to a competition factor (CF) of 1. CF values greater than 1 are characterized by a lower affinity for the progesterone receptor, and CF values of less than 1 are characterized by higher affinity.
Binding Assay: Using cytosol from progesterone receptor-expressing insect cells (Hi5), competitive binding to the progesterone receptor was determined from the ability to displace 3H-progesterone as reference substance from the receptor. If a compound has an affinity corresponding to progesterone, this corresponds to a competition factor (CF) of 1. CF values greater than 1 are characterized by a lower affinity for the progesterone receptor, and CF values of less than 1 are characterized by higher affinity.
Inhibition Assay: To conduct the assays, the 250 uM monasuspiloin solution was further diluted with 10% DMSO to prepare 25 uM, and 5 uM monasuspiloin samples, and the final concentration of DMSO in the cell culture was 1%.The cell line used in glucocorticoid receptor (GR) assay was human A-549 cells (cultured in Ham's F12K medium (Gibco, USA), 10% FBS), the cell line used in progesterone receptor (PR) assay was human T-47D cells (cultured in RPMI 1640 medium (Gibco, USA), 10% FBS), and the cell line used in estrogen receptor (ER) assay was human MCF7 cells (cultured in MEM medium (Gibco, USA), 10% FBS). The cells in the exponential phase of growth were washed off with 1 ml of 0.05% trypsin, centrifuged and collected in a centrifuge tube. 1x107 cells were resuspended in 270 ul BES medium (5 mM BES in medium) and 8 to 10 ug of plasmid [pMMTV-SEAP plasmid was used for GR and PR (see FIG. 7), and pTA-ERE-SEAP plasmid (Clontech, USA) was used for ER (see FIG. 8)] were also added.