Investigating RXR dependent changes in mRNA expression by using novel chemical modulators
The ligand dependent nuclear receptor subtypes RXRa, RXRb, and RXRc are key mediators in mammalian homestasis and in response to pathological conditions, e.g. inflammation and neurodegeneration. We allocated a set of RXR pan-agonists including the established agonist Bexarotene, a drug used as second line treatment for cutaneous T-cell lymphoma, and four additional chemical tools (JP147, JP175, PH299, S169), that surpass Bexarotene in terms of physicochemical properties and - more importantly - selectivity within the family of nuclear receptors.  The ligand dependent nuclear receptor subtypes RXRa, RXRb, and RXRc are key mediators in mammalian homestasis and in response to pathological conditions, e.g. inflammation and neurodegeneration. We allocated a set of RXR pan-agonists including the established agonist Bexarotene, a drug used as second line treatment for cutaneous T-cell lymphoma, and four additional chemical tools (JP147, JP175, PH299, S169), that surpass Bexarotene in terms of physicochemical properties and - more importantly - selectivity within the family of nuclear receptors. We investigated changes in RNA expression profiles in HEK293T cells upon stimulation with said modulatirs. HEK293T cells were seeded at a density of 3 x 10^6 cells per well in 12-well plates and complete culture medium. After 24 h, medium was changed to DMEM high glucose supplemented with sodium pyruvate (1 mM), penicillin-streptomycin (100 U/mL and 100 µg/mL, respectively) and 0.2% FCS for synchronization over 24 h. Medium was changed again to DMEM high glucose supplemented with sodium pyruvate (1 mM), penicillin-streptomycin (100 U/mL and 100 µg/mL, respectively) and 0.2% FCS, additionally containing either test compounds  (1 µM) in 0.1% DMSO or 0.1% DMSO. After 17 h, cells were harvested and total RNA was isolated using E.Z.N.A. Total RNA Kit I (Omega Bio-tek) according to the manufacturer's instructions. RNA concentration and purity were assessed using a NanoDrop One UV–vis spectrophotometer (Thermo Fisher Scientific) at 260/280 nm. We isolated total RNA from ~ 300.000 cells per sample using silica gel columns and submitted the samples to RNA sequencing at Novogene (United Kingdom).