Cross-talk of RORA and NPSR1 in cell models.
(A) Real-time PCR analysis of circadian clock genes in human SH-SY5Y neuroblastoma cell line over-expressing NPSR1 stimulated with 100 nM NPS for 0–24 h in the presence or absence of 3 µM SHA 68 (N-[(4-fluorophenyl)methyl]tetrahydro-3-oxo-1,1-diphenyl-3H-oxazolo[3,4-a]pyrazine-7(1H)-carboxamide), a selective antagonist of NPSR1. (B) Real-time PCR analysis of RORA, NR1D1, NPAS2, CLOCK, ARNTL, PER1, CRY1, and DBP mRNA expression in human embryonic kidney epithelial HEK-293H cell line over-expressing NPSR1 6 h after NPS (0.1–5 µM) stimulation. The results are presented as fold-changes in comparison to the unstimulated cells. GAPDH was used as the endogenous reference, and data are expressed as mean of triplicate samples ±95% confidence intervals. In all experiments, results were calculated with the comparative ΔΔCt method. (C) Schema of the NPSR1 promoter and the location of the putative 6-bp AT-rich sequence preceding the half-core motif PuGGTCA (RORE). (D) NPSR1 driven luciferase in relative luminescence units after transfection with either RORA-1 encoding plasmid or an empty vector. Ten biological replicates per group. Mann-Whitney U test ***p<0.0001.
