Nurr1

DA neuronal cultures were immunostained with antibodies against various dopaminergic neuronal markers as described below. DA neuronal cultures were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature (RT) and washed with PBS and permeabilized with 0.1% Triton‐X in PBS for 5 min before blocking for 1 h in 10% goat or donkey serum in PBS. Cells were then incubated with primary antibodies: TH (Rabbit polyclonal, Millipore 1:1000, Billerica, MA), TUJ‐1 (Mouse monoclonal, Millipore, 1:1000 Billerica, MA), VMAT2 (Rabbit polyclonal, Millipore, 1:1000, Billerica, MA), Nurr1 (Goat polyclonal, R&D, 1:500 Minneapolis, MN) diluted in 10% serum in PBS at 4°C overnight. After three washes in PBS, the cells were incubated with fluorescent secondary antibodies at RT for 1 h. Cells were counterstained with DAPI and images were acquired using an Olympus microscope (Olympus, Center Valley, PA). Omission of the primary or secondary antibodies resulted in no staining and served as negative controls. Percentage of TH‐positive cells and percentage of TUJ‐1‐positive cells were quantified using Nikon NIS‐Elements software and averaged from 10 randomly selected fields in each culture well. The percentage was determined in 6–9 wells of cells for each line pooled from three independent experiments and the SEM refers to the standard errors among these 6–9 wells for each line.

Azoramide (Cat# SYA965090) was purchased from Toronto Research Chemicals. For immunostaining, DAPI (Cat# C1005) was obtained from Beyotime. LMX1A (Cat# HPA030088) was from Atlas. FOXA (Cat# 22474-1-AP) was from Proteintech. DAT (Cat# MAB369) was from Millipore. TH (Cat# P40101-150) was obtained from Pel Freez. Girk2 (Cat# APC-006) was obtained from Alomone Laboratories. Nurr1 (Cat# PP-N1404-00) was from R&D. MAP2 (Cat# 4542S) was from Cell Signaling Technology. TUJ1 (Cat# T8578) was obtained from Sigma. For Western blotting, cleaved caspase 3 (Cat# 9961S), cytochrome C (Ca# 11940), Bax (Cat# 2772S), Bcl-2 (Cat# 2870S), PERK (Cat# 3192), Drp-1 (Cat# 8570S), Mfn-1 (Cat# 14739S), CHOP (Cat# 2895) and CREB (Cat# 9197S) were purchased from Cell Signaling Technology. Fis-1 (Cat# PA5-22142) was from Invitrogen. Bip (Cat# ab21685) was obtained from Abcam.

Prior to proceeding with the IHC/IF protocol, a standard process of deparaffinizing sections was performed in xylol and alcohol series. Antigen retrieval was performed by boiling sections in citrate buffer (pH 6,0). After three washes in PBS, blocking solution containing 1–3% BSA and 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MI, USA) in PBS was applied on the sections for 1–2 h. The blocking solution was replaced with primary antibodies diluted in a blocking solution and incubated overnight at 4 °C. The following antibodies were used: NeuN (Abcam, Cambridge, UK), MAP2 (Sigma-Aldrich, St. Louis, MI, USA), MAP2 (Merck Millipore, Burlington, MA, USA), CUX2 (Abnova, Taipei, Taiwan), CUX2 (Abcam, Cambridge, UK), Reelin (Millipore), DCX (Merck Millipore, Burlington, MA, USA), FOXP1 (Abcam, Cambridge, UK), Neuroserpin (Abcam, Cambridge, UK), TLE4 (Santa Cruz Biotechnology, Dallas, TX, USA), Nurr1 (R&D systems, Minneapolis, MN, USA). After incubation, sections were washed in PBS, and appropriate secondary antibodies (Alexa Fluor, Thermo Fisher Scientific, Waltham, MA, USA) were applied for 2 h at RT. Following three washes in PBS, TrueBlack quencher (Biotium, Fremont, CA, USA) was applied on the sections. Finally, the sections were covered using a mounting medium with DAPI (Vectorlabs, Burlingame, CA, USA). The IHC protocol was implemented as previously described [32 (link)].