Gly-Ser linker. The resulting plasmid was termed pFTI-CMV (fusion trans-inducing factor plasmid). Gal4-TLX/Gal4-VP16 assay procedure. HEK293T cells were grown in Dulbecco´s modified Eagle´s medium (DMEM, ThermoFisher Scientific), high glucose with 10% fetal calf serum (FCS), sodium pyruvate (1 mM), penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37 °C and 5% CO2. 24 hours before transfection, cells were seeded in 96-well plates (30,000 cells/well) in DMEM with above mentioned supplements. Prior to transfection, medium was changed to Opti-MEM (ThermoFisher Scientific) without supplements. Cells were then transiently transfected with plasmid mixtures containing 100 ng/well pFR-Luc, 1 ng/well pRL-SV40, 6 ng/well of pECE-SV40-Gal4-VP16 and 3 ng/well of pFA-CMV-Gal4-TLX (during assay establishment, these plasmid amounts per well were systematically varied to optimize conditions allowing robust observation of bidirectional TLX modulation). Transient transfection was achieved using Lipofectamine LTX reagent (ThermoFisher Scientific) according to the manufacturer´s protocol. Five hours after transfection cells were treated with Opti-MEM supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL) additionally containing 0.1% dimethylsulfoxide (DMSO) and the respective test compounds or 0.1% DMSO alone as negative control. Each sample was tested in duplicates and every experiment was conducted at least three times. After 14 h incubation, cells were lysed for luciferase luminescence detection using the Dual-Glo Luciferase Assay System and XhoI. The sequence coding for the RXRα LBD followed by a stop codon was then introduced in frame between the afore inserted restriction site for BamHI and XhoI. For expression of TLX LBD with N-terminal GFP the sequence encoding TLX residues 150-385 was cloned between the same sites. For generation of biotinylated TLX LBD, the pMal vector system (New England Biolabs, NEB, Ipswich, MA, USA) was used. In pMal-c2E, the section between the sequence encoding 10x Asparagine (Asn10) and the SalI restriction site was replaced with a sequence encoding Leu-Gly-Ile-Glu-Leu-Val-[His8-Tag]-Asp-Tyr-Asp-Ile-Pro-Gly-Thr-Leu-[TEV site]