Secosteroid Transcriptional Activity Assay

Caco-2, HaCaT and Jurkat cells were cultured as described previously^{1 (link),4 (link),23 (link),32 (link)}, 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). After one week selection by puromycin (1 µg/mL), cells were seeded in a 96-well plate (10,000 cells/well with a volume of 100 µL/well) using FBS-free medium and synchronized for 24 h. DMSO solutions (1 µL) of secosteroids to be tested were added to cells, which were then incubated for another 24 h. The luciferase signal was then measured according to the manufacturer’s procedure for the ONE-Glo^TM Luciferase Assay System (Promega, Madison, WI, USA). The final concentration of DMSO was 0.1% and 0.1% DMSO was used as the vehicle control. All concentrations were tested in triplicate.