Sh sy5y cells

SH-SY5Y cells were purchased from American Type Culture Collection (ATCC, USA) and cultured in Dulbecco's modified Eagle medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated horse serum (Gibco), 5% heat-inactivated fetal calf serum (Gibco), 100 IU/ml penicillin, and 100 mg/ml streptomycin in a humidified incubator containing 5% CO₂ at 37°C. SH-SY5Y cells were treated with 50 ng/ml 2.5S nerve growth factor (Promega, Madison, WI, USA) for 9 days to induce differentiation. Cells were plated in 6-well plates and passaged at 60–70% confluence. In the experiments, cells were pretreated with 25–100 μM Sal for 24 h and then exposed to 500 μM MPP+ for an additional 24 h. In some experiments, cells were transfected with siNrf2 or siDJ-1 (Santa Cruz Biotechnology) using lipofectamine 2000 transfection reagent 2–4 days before the treatment of Sal and MPP+.

Human neuroblastoma SH-SY5Y cells (ATCC, Manassas, VA) were grown in DMEM containing 10% FBS (vol/vol) and antibiotics in a humidified 5% CO2/95% air atmosphere at 37°C. Adipose derived mesenchymal stromal cells (hAD-MSCs) [41 ] were grown in Mesenchymal Stem cell Expansion medium (Millipore, Billerica, MA, USA), and the culture media was replaced every 3 days. X-tremeGENE HP transfection reagent (Roche) was used for transient transfections according to the manufacturer's instructions. Unless otherwise indicated, lysates were prepared 48 h after transfection. For the luciferase assay, SH-SY5Y cells were transiently transfected with pGL3-Basic, pGL3-PGC-1α promoter-Luciferase, CRE deletion mutant luciferase constructs for firefly Luciferase assay, and 10 ng pRL-TK vector (Promega) for Renilla luciferase control.

Human neuroblastoma SH-SY5Y cells (Sigma-Aldrich, Merck, Kenilworth, NJ) were cultured in Eagle’s Minimum Essential Medium and Ham’s F12 (1:1) (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (FBS), glutamine and antibiotics (1% streptomycin and penicillin) (all from Gibco, Thermo Fisher Scientific), and cultivated in T25 flasks at 37°C with 5% (v/v) CO₂ at saturated humidity. SH-SY5Y cells were stably transfected with a plasmid construct, pCI-neo (Promega, Madison, WI) encoding human PRNP, using jetPRIME (Polyplus, Illkirch, France) according to the manufacturer's instructions. Transfected cells were grown under selection pressure of Geneticin (Thermo Fisher Scientific), and nine different single clones with variable levels of PrP^C (SH-SY5Y PrP^high) were isolated (S4 Fig). Clone no. 8 showed an abnormal phenotype, and was excluded from the studies.