Transient transactivation assay

Full-length VDR constructs were tested in transient transactivation assays with 1α, 25-dihydroxyvitamin D₃ (1, 25D3) (EMD Millipore, Billerica, MA) as the positive control. Experiments were conducted using pSG5hVDR, pRLCMV, and CYP24-Luc expression vectors as previously described⁵⁹. Hek293T cells were seeded in 96-well plates at 2.5 × 10⁴ cells per well 24 hours prior to transfection. Cells were transfected at 90–95% confluency using Lipofectamine 2000 (Life Technologies, Grand Island, NY) with DNA diluted in Opti-MEM I Reduced Serum Medium as per the manufacture’s recommendations. For functional comparisons, 89.7 ng of each pSG5-VDR construct was transiently transfected into Hek293T cells with 19.2 ng CYP24-Luc and 4.5 ng of Renilla luciferase, which serves as an internal luciferase control (Promega Corporation, Madison, WI). The media was changed 18 hours post transfection and cells were exposed to compounds in fresh DMEM media. Twenty-four hours post-exposure to compounds the Dual-Glo Luciferase Assay System (Promega Corporation, Madison, WI) was used to passively lyse the cells and test for luciferase activity following the manufacturer’s protocols. Luciferase activities were measured using a Wallac MicroBet TriLuc Luminometer (Perkin Elmer Life Sciences, Waltham, MA). Control reactions included empty pSG5 vector and ethanol as a vehicle control. Luciferase readings were normalized to the internal Renilla control, and VDR response was normalized to an empty vector control. Transient transfection data were used with the ToxCast Analysis Pipeline (TCPL) to generate dose-response curves and estimate AC₅₀ values^55,57. Curve fitting was performed using Hill, Gain Loss (GNLS), and constant fit models. AC₅₀ values were generated for antagonists or agonists, respectively, and were chosen from either the Hill or GNLS model using the Akaike Information Criterion.