*4.1. Reagents and Antibodies*

1-methyl-4-phenylpyridinium ion MPP<sup>+</sup>, H2O2, and 3-(3,4-dimehylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 96-well tissue culture plates, along with six-well and 100 mm culture dishes, were obtained from Nunc Inc. (North Aurora Road, Naperville, IL, USA). Foetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM/F12) were purchased from Gibco-BRL Technologies (Gaithersburg, MD, USA). RIPA buffer (10×) was purchased from Millipore (Milford, MA, USA). Tween 80 was obtained from Calbiochem (Gibbstown, NJ, USA). All other chemicals utilised in this research were of analytical grade and were purchased, unless otherwise noted, from Sigma-Aldrich.

#### *4.2. Cell Culture and Transfection*

The human dopaminergic neuroblastoma SH-SY5Y cell line was acquired from the American Type Culture Collection (ATCC; Manassas, VA, USA). SH-SY5Y cells were cultured in DMEM/F12 with or without phenol red and HEPES, supplemented with 100 U/mL penicillin/streptomycin and 10% (v/v) inactivated foetal bovine serum. The SH-SY5Y cells were maintained in a 5% CO2 and 95% humidified air incubator at 37 ◦C for the time indicated in the experiments. MPP<sup>+</sup> and H2O2 were dissolved in three-times distilled water (3DW).

To overexpress and knockout the human GPR4 gene, a GPR4 lentiviral vector (CMV; pLenti-GIII-CMV-C-term-HA) and a GPR4 sgRNA CRISPR/Cas9 lentiviral vector (pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro) were designed, to generate stable GPR4-overexpressing (GPR4-OE) and stable GPR4-knockout (GPR4-KO) SH-SY5Y lines. Briefly, the SH-SY5Y cells were transferred into 60 mm plates at a density of 5 <sup>×</sup> 104 cells/mL to achieve ~70% confluence at the time of transfection. The cells were transfected using the Lipofectamine® 3000 transfection reagent (ThermoFisher, Langenselbold Germany; #L3000015), according to the manufacturer's protocol. Both the GPR4-overexpression and knockout-silencing genes were designed to carry puromycin-resistance genes and were produced using the Lentivector Expression System (Applied Biological Materials Inc. (ABM), Canada). Stable single clones were selected following 3–5 weeks of puromycin treatment (1 μg/μL). GPR4 overexpression and knockout in the stably infected clones were assessed through RT-PCR and western blotting. A sequence analysis of the GPR4 insert was also employed.
