Cho tet on cells

Doxycycline-inducible ROR stable cell lines were generated by transfecting a pTRE2 expression vector (Clontech, Mountain View, CA, USA) containing RORα or RORγ, into CHO Tet-on cells (Clontech) followed by transfection with the pGL4.27 luciferase (LUC) reporter vector (Promega, Madison, WI, USA) driven by 5xRORE. Single clones of pGL4.27-5xRORE- and pTRE2-ROR-expressing cells were selected from a medium containing hygromycin (Invitrogen, Grand Island, NY, USA) and puromycin (Sigma–Aldrich), respectively. CHO Tet-on cell lines were cultured in F12 medium supplemented with 10% FBS, and suitable for use in the Tet-on system (Clontech). To induce ROR expression, cells were treated for 20 h with 1 μM doxycycline in the presence or absence of a dilution series of the isoflavones. All the assays were performed in triplicate and repeated independently at least twice. RORE-mediated activation of the LUC reporter was measured with a Luciferase Assay Substrate Kit (Promega). cAMP-based cell viability was evaluated by CellTiter-Glo Luminescent Cell Viability Assay (Promega).