CSLs Agonize RORα and Inhibit RORγ Transcription: A Dual Luciferase Reporter System Study

We utilized a dual luciferase reporter system to investigate the impact of CSLs on RORα and RORγ. Initially, the GAL4 transcription factor, produced by the fusion gene, specifically recognizes the cis-acting element on plasmid pG5, which initiates the expression of the downstream FLuc reporter gene and normalizes the luciferase values using RLuc. Based on this principle, the drug intervention results are displayed in Fig. 10. After conducting CCK8 analysis and calculating the IC50 for CSL (IC50=6.44 μmol/L) (Fig. 9), we selected a drug concentration of 2 μmol/L for the ROR-LBD intervention. Using Dimethyl sulfoxide (DMSO) as the control, CSLs agonized RORα (P < 0.01), with 18-OH-CSL and 28-OH-CSL demonstrating maximum potency, and an increase of approximately 40% in RORα transcription, as depicted in Fig. 10A. RORγ transcriptional activity was inhibited (p < 0.01), with 12-OH-CSL, a-ring aromatized 15-OH-CSL demonstrating maximum potency, and an approximately 30% reduction in RORγ transcription, as shown in Fig. 10B. After combining the compound with RORα binding site amino acids GLN19, ARG97, ARG100 (Fig. 10C) and RORγ binding site, following mutation of amino acids GLN25, LEU26, ARG103, ARG106 (Fig. 10D), transfection experiments illustrated that CSLs had almost no further effect on RORs.