Full-Length Nurr1 Reporter Gene Assays. Plasmids: The reporter plasmids pFR-Luc-NBRE , pFR-Luc-NurRE , and pFRLuc-DR5 each containing one copy of the respective human Nurr1 response element NBRE Nl3 (TGATATCGAAAACAAAAGGTCA), NurRE (from POMC; TGATATTTACCTCCAAATGCCA), or DR5 (TGATAGGTTCACCGAAAGGTCA) were described previously. The full length human nuclear receptor Nurr1 (pcDNA3.1-hNurr1-NE; Addgene, entry 102363) and, for DR5, RXRα (pSG5-hRXR ) were overexpressed. pFL-SV40 (Promega) was used for normalization of transfection efficiency and evaluation of compound toxicity. Assay procedure: HEK293T cells were grown in DMEM high glucose, supplemented with 10% FCS, sodium pyruvate (1 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C and 5% CO2. The day before transfection, HEK293T cells were seeded in 96-well plates (3 × 10 4 cells/well). The medium was changed to Opti-MEM without supplements right before transfection. Transient transfection was performed using the Lipofectamine LTX reagent (Invitrogen) according to the manufacturer's protocol with pFR-Luc-NBRE , pFR-Luc-NurRE or pFR-Luc-DR5 , pRL-SV40 (Promega), the human full length receptor plasmid pcDNA3.1-hNurr1-NE, and, for DR5, also pSG5-hRXR . Five hours after transfection, the medium was changed to Opti-MEM supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL), now additionally containing 0.1% DMSO and the respective test compound or 0.1% DMSO alone as untreated control. Each sample was tested in duplicates and each experiment was performed independently three times. Following 16 h incubation with the test compounds, the cells were assayed for luciferase activity using the Dual-Glo TM Luciferase Assay System (Promega) according to the manufacturer's protocol. Luminescence was measured with a Spark 10 M luminometer (Tecan Group AG). Normalization of transfection efficiency and cell growth were done by division of firefly luciferase data by renilla luciferase data and multiplying the value by 1000 resulting in RLU. Fold activation was obtained by dividing the mean RLU of a test compound at a respective concentration by the mean RLU of untreated control. The full length Nurr1 reporter gene assays were validated with amodiaquine (2) and chloroquine as reference agonists.