Cignal lenti vdre reporter luc kit

HaCaT and Caco-2 cells were transduced with lentiviral VDRE luciferase using a Cignal Lenti VDRE reporter (luc) kit according to the manufacturer’s protocol (QIAGEN, Valencia, CA). After 1 week selection by puromycin (1 µg/mL), transduced HaCaT and Caco-2 cells were seeded in a 96-well plate (1000 cells/well) using partial media (without FBS) and incubated for 24 h. DMSO solutions of secosteroids to be tested were added to cells, which were then incubated for another 24 h, and the luciferase signal was measured according to the manufacturer’s procedure for the ONE-Glo luciferase assay system (Promega, Madison, WI). The final concentration of DMSO was 0.1%, and 0.1% DMSO was used as the vehicle control. All concentrations were tested in triplicate.

Jurkat cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and were transduced with lentiviral VDRE luciferase using a Cignal Lenti VDRE Reporter (luc) Kit according to the manufacturer’s protocol (QIAGEN, Valencia, CA, USA). The cells then went through 1-week selection under puromycin (1.0 μg/ml) treatment. During the selection, the media were changed every other day. For the biological test, transduced Jurkat cells were washed with PBS (1×) and then seeded in a 96-well plate (10,000 cells/well, 100 μl/well) diluted by FBS-free media. All cells were then synchronized by a 24 h incubation. DMSO solutions (10%; 1.0 μl) of each secosteroid were added to each well of cells with final concentrations of 1000, 300, 100, 30, 10, 3 and 1 nM, which were then incubated for another 24 h. The luciferase signal of the cells was measured by the ONE-Glo™ Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer’s suggested procedure. DMSO (10%) in water was used as the vehicle control and the final concentration of DMSO in culture media was 0.1% during the activity test. Each concentration of analog was tested in triplicate (n=3).