Ectopic DRD2 and DRD3 expression

HEK293 cells were cultured in Dulbecco´s Modified Eagle´s Medium (DMEM; Biowest, L104) supplemented with 10% FBS and 1% penicillin-streptomycin. Cultures were maintained in a humidified incubator set at 37ºC and 5% CO₂. Semiconfluent cells were transfected with pCEP4-EGFP-DRD3- or pcDNA3.1+/Hygro-GFP-DRD2- using Lipofectamine 2000 (Thermo Fisher Scientific, 11,668–027) at a ratio of 2:1 according to the manufacturer´s protocol. The plasmids were a gift from Dr. Jean-Michel Arrang (Addegene, 24,098 and 24,099 [⁸⁸]). Stable cell lines expressing EGFP-DRD3- and GFP-DRD2- were obtained by growth in selective medium containing 200 μg/ml hygromycin B (Thermo Fisher Scientific, 1068–7010). After 8–10 d of selection in G418 (Sigma-Aldrich, A1780), individual clones were expanded in multi-well plates and examined for expression by western-blot and immunofluorescence for GFP. Several positive clones were identified and used for subsequent experiments. Subconfluent cell cultures were grown and treated with PPX as indicated in the text and figure legends. In some cases, the autophagy inhibitor chloroquine diphosphate (CQ, 20 μM, 30 min before PPX), or the MEK/MAPK1/3 inhibitor PD98059 (25 μM, 1 h; Cell Signaling Technology, 99,005) were added to the medium. Each experiment was performed in triplicate.