*2.1. Expression of GPR4 Is Upregulated in Neurotoxin-Stimulated Apoptosis in SH-SY5Y Cells*

To investigate the concentrations of MPP<sup>+</sup> and H2O2 that precipitated a cell death of nearly 50% in the SH-SY5Y cells, 24 h serum-starved SH-SY5Y cells were treated with MPP<sup>+</sup> (0.25, 0.5, and 1 mM) or H2O2 (50, 75, and 125 μM) for 24 h. As is shown in Figure 1A, when treated with the various concentrations of MPP<sup>+</sup> (1 mM; 56.511 <sup>±</sup> 1.55%) and H2O2 (125 <sup>μ</sup>M; 53.12 <sup>±</sup> 2.34%), half of the cell population in the MTT assay died. Furthermore, the mRNA and protein expressions of GPR4 in SH-SY5Y cells in both MPP<sup>+</sup>- (1 mM) and H2O2- (125 μM) treated serum-free media gradually increased in a time-dependent manner (3–24 h; Figure 1B).

**Figure 1.** The cellular viability and the mRNA and protein expressions of GPR4 in MPP+ and H2O2-treated SH-SY5Y cells. 24 h serum-starved SH-SY5Y cells were treated with different concentrations of MPP<sup>+</sup> (0.25, 0.5, and 1 mM) and H2O2 (50, 75, and 125 μM) for 24 h in serum-free culture media. (**A**) The cellular viability of the SH-SY5Y cell after treatment with different concentrations of MPP<sup>+</sup> and H2O2 in serum-free media for 24 h. (**B**) Reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting demonstrate the mRNA and protein expressions of GPR4 in SH-SY5Y cells at different time points (3, 6, 12, 18, and 24 h) after stimulation with MPP<sup>+</sup> (1 mM) and H2O2 (125 μM) in serum-free media. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were utilised as the internal controls. Mean ± standard error of the mean (SEM; *n* = 3) was employed to express the data. Tukey's multiple comparison test was performed using a one-way analysis of variance (ANOVA). Each \* *p* < 0.05 refers to the other sample concentrations compared with the control cells.
