*2.3. Knockout of GPR4 Decreases the Bax*/*Bcl-2 mRNA Ratio during Neurotoxin-Induced Apoptosis in SH-SY5Y Cells*

To determine the role of GPR4 in both MPP<sup>+</sup>- (1 mM) and H2O2- (125 μM) stimulated apoptotic cell death, we investigated the expression levels of the Bcl-2 family proteins (Bax and Bcl-2). Many studies suggest that the Bcl-2 family plays a critical role in the mitochondrial apoptotic pathway. Bax enhances the release of cytochrome C from the space of the mitochondrial intermembrane to the cytosol, resulting in apoptosis. In contrast, Bcl-2 prevents apoptosis through its prevention of cytochrome C release, thereby maintaining mitochondrial cellular integrity [29,30]. In this study, an RT-PCR was employed to assess the mRNA expression levels of GPR4, Bax, and Bcl-2 in 24 h serum-starved SH-SY5Y cells treated with either MPP<sup>+</sup> (1 mM) or H2O2 (125 μM; Figure 3A).

**Figure 3.** The measurement of GPR4 mRNA expression and the Bax/Bcl-2 mRNA ratio in MPP+- and H2O2-treated SH-SY5Y cells that were stably GPR4-OE or GPR4-KO. (**A**) An RT-PCR illustrating the mRNA expression of pro-apoptotic Bax, anti-apoptotic Bcl-2, and GAPDH in SH-SY5Y, GPR4-OE, and GPR4-KO cells after stimulation with MPP<sup>+</sup> (1 mM) and H2O2 (125 μM) in serum-free media for 24 h. (**B**) A semi-quantification of the GPR4 mRNA and Bax/Bcl-2 mRNA expressions relative to GAPDH. This semi-quantification of the respective mRNA expression levels was performed on ImageJ software; GAPDH was utilised as an internal control. 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 sample concentration compared with the same group of non-treated cells.

A semiquantitative analysis (Figure 3B) of the RT-PCR bands highlighted a more than 4-fold increase in the expression of GPR4 in the GPR4-OE cells without any treatment, compared with the non-treated SH-SY5Y cells. In comparison with the non-treated SH-SY5Y cells, neurotoxins increased the expression of GPR4 in the GPR4-OE cells by 3–4-fold (MPP+, 3.65 <sup>±</sup> 0.03; H2O2, 3.31 <sup>±</sup> 0.17), whereas no significant difference in the GPR4 expression of the GPR4-OE cells (MPP+, 0.53 <sup>±</sup> 0.003; H2O2, 0.04 ± 0.003) was observed.

Interestingly, the ratio of Bax/Bcl-2 mRNA expression for non-treated GPR4-OE cells was slightly higher (1.40 ± 0.08) than that for the control SH-SY5Y cells, whereas the Bax/Bcl-2 mRNA ratio was slightly lower (0.73 ± 0.904) in the non-treated GPR4-KO group than that for the control SH-SY5Y cells. For the SHSY-5Y cells, treatment with MPP<sup>+</sup> or H2O2 significantly increased the ratio of Bax/Bcl-2 mRNA expression (MPP+, 1.59 <sup>±</sup> 0.02; H2O2, 1.55 <sup>±</sup> 0.04). Yet, the ratio of Bax/Bcl-2 mRNA expression in MPP<sup>+</sup>- and H2O2-stimulated GPR4-OE cells was significantly higher (MPP<sup>+</sup>, 1.92 <sup>±</sup> 0.03; H2O2, 1.88 ± 0.07) than the neurotoxin-treated SH-SY5Y and non-treated GPR4-OE cells. Meanwhile, the ratio of Bax/Bcl-2 mRNA expression in both MPP<sup>+</sup>- and H2O2-stimulated GPR4-KO cells was significantly lower (MPP+, 1.38 <sup>±</sup> 0.02; H2O2, 1.25 <sup>±</sup> 0.02) than both the neurotoxin-treated SH-SY5Y and GPR4-OE cells.
