*2.5. Knockout of GPR4 Decreases the Bax*/*Bcl-2 Protein Ratio and the Cleavage of PARP Expression in Neurotoxin-Stimulated SH-SY5Y Cells*

MPP+-treated SH-SY5Y cells were assessed with immunoblotting to evaluate the effect of GPR4 antagonist, NE52-QQ57, on GPR4; the pro-apoptotic proteins, Bax and Bcl-2; and cleaved PARP expression (Figure 5). The SH-SY5Y cells were pre-treated with NE52-QQ57 (100 nM) at pH 7.4. This was followed by 24 h incubation, or MPP<sup>+</sup> (1 mM) stimulation for 24 h in serum-free media.

**Figure 5.** The effect of GPR4 antagonist, NE52-QQ57, on the GPR4 and pro-apoptotic protein expressions in MPP+-treated SH-SY5Y cells that were stably GPR4-OE or GPR4-KO. 24 h serum-starved SH-SY5Y cells were pre-treated for 1 h with NE52-QQ57 (100 μM); this was followed by MPP<sup>+</sup> (1 mM) stimulation for 24 h in serum-free culture media. (**A**) An immunoblot and semi-quantification of the respective protein expressions of GPR4, Bax, Bcl-2, Cleaved PARP, and β-Actin in SH-SY5Y cells. (**B**) An immunoblot and semi-quantification of the respective protein expressions of GPR4, Bax, Bcl-2, cleaved PARP, and β-Actin in GPR4-OE cells. (**C**) An immunoblot and semi-quantification of the respective protein expressions of GPR4, Bax, Bcl-2, cleaved PARP, and β-Actin in GPR4-KO cells. β-Actin 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.

For the control SH-SY5Y cells, MPP<sup>+</sup> stimulation significantly increased the Bax/Bcl-2 ratio (22.94 ± 2.02 folds) and the cleaved PARP (5.09 ± 0.18 folds), in comparison with the non-treated control SH-SY5Y cells. Pre-treatment with the NE52-QQ57 (100 nM) significantly lowered the GPR4 expression (3.21 ± 0.18 folds), Bax/Bcl-2 ratio (16.47 ± 1.45 folds), and cleavage of PARP (4.29 ± 0.15 folds) in the MPP+-stimulated cells, in comparison with the SH-SY5Y cells that were only MPP+-treated (Figure 5A). For GPR4-OE cells, MPP<sup>+</sup> stimulation significantly increased the Bax/Bcl-2 ratio (29.49 <sup>±</sup> 2.06 folds) and the cleaved PARP (6.85 ± 0.23 folds), in comparison with the non-treated GPR4-OE cells. Similar to the RT-PCR results, pre-treatment with NE52-QQ57 (100 nM) significantly lowered the GPR4 expression (0.13 ± 0.003 folds), Bax/Bcl-2 ratio (17.81 ± 0.86 folds), and cleavage of PARP (4.98 ± 0.26 folds) in the MPP+- and NE52-QQ57-treated cells, in comparison with the GPR4-OE cells that were only treated with MPP<sup>+</sup> (Figure 5B). In the GPR4-KO cells, MPP<sup>+</sup> increased the Bax/Bcl-2 ratio (19.15 <sup>±</sup> 1.45 folds) and cleaved PARP (4.99 ± 0.27 folds), in comparison with non-treated GPR4-KO cells. In contrast, MPP+-stimulated GPR4-KO cells that were pre-treated with NE52-QQ57 (100 nM) demonstrated only a minor increase in Bax/Bcl-2 ratio (15.24 ± 0.91 folds) and cleavage of PARP (4.23 ± 0.22 folds), in comparison with GPR4-KO cells that were only MPP+-treated (Figure 5C). Overall, the GPR4-KO

cells displayed a lesser increase in the Bax/Bcl-2 ratio (19.15 ± 1.45 folds) and a decrease in the PARP cleavage (4.99 <sup>±</sup> 0.27 folds), in comparison with both the MPP+-stimulated SHSY-5Y (Bax/Bcl-2, 22.94 ± 2.02 folds; cleaved PARP, 5.09 ± 0.18 folds) and GPR4-OE cells (Bax/Bcl-2, 29.49 ± 2.06 folds; cleaved PARP, 6.85 <sup>±</sup> 0.23 folds). Thus, less apoptotic cell death was induced by MPP+.
