*2.2. Knockout of GPR4 Protects SH-SY5Y Cells from Neurotoxin-Stimulated Apoptosis in SH-SY5Y Cells*

To assess the effect of GPR4 overexpression and knockout on MPP+-induced apoptotic cell death, 24 h serum-starved SH-SY5Y cells were treated with MPP<sup>+</sup> (1 mM) for 24 h in serum-free media

(Figure 2). Following the MPP<sup>+</sup> (1 mM) treatment for 24 h in serum-free media, the number of SH-SY5Y viable cells decreased. Furthermore, the cells became rounded, displayed an increased neurite retraction, and were found to be loosely attached to the plate. Under bright-field optics, the GPR4-OE cells treated with MPP<sup>+</sup> (1 mM) exhibited less cell viability, with increased rounded cells, increased neurite retraction, and loose attachment to the surface. In contrast, the GPR4-KO cells treated with MPP<sup>+</sup> (1 mM) were more viable, strongly attached, neuronal shaped, and demonstrated less neuronal retraction than both the control and the GPR4-OE cells (Figure 2A).

**Figure 2.** The cellular viability and morphology of MPP+-treated SH-SY5Y cells that were stably GPR4-overexpressing (GPR4-OE) or GPR4-knockout (GPR4-KO). 24 h serum-starved SH-SY5Y cells were treated with MPP<sup>+</sup> (1 mM) for 24 h in serum-free culture media. (**A**) The morphology of SH-SY5Y GPR4-OE and GPR4-KO cells was observed through bright-field microscopy. (**B**) Cell viability was evaluated using an MTT assay. Mean ± SEM (*n* = 3) was employed to express the data. Tukey's multiple comparison test was performed using a one-way ANOVA. Each \* *p* < 0.05 refers to the other sample concentrations compared with the control cells.

Cell viability was assessed with an MTT assay. The control SH-SY5Y cells presented a 55.67 ± 5.22% cell survival rate, whereas only 42.00 <sup>±</sup> 2.01% of the GPR4-OE cells treated with MPP<sup>+</sup> (1 mM) survived. In contrast, the MPP+-treated GPR4-KO cells had a significantly higher cell survival rate (71.63 <sup>±</sup> 3.54%), at 15% higher than for the MPP+-treated control SH-SY5Y cells and almost 30% higher than for the MPP+-treated GPR4-OE cells (Figure 2B).
