*2.4. E*ff*ect of a GPR4 Antagonist on the Cellular Morphology and GPR4 mRNA Expression of SH-SY5Y Cells*

To investigate the effect of the pharmacological inhibition of GPR4, we adopted a GPR4 antagonist, NE52-QQ57. At the physiological pH, NE 52-QQ57 has been reported to effectively block the cAMP that is released by GPR4 activation (IC50 26.8 nM) in HEK293 cells [3]. In this study, the impact of GPR4

antagonist, NE52-QQ57, on the cellular morphology and mRNA expression of GPR4 in MPP+- (1 mM) treated, 24 h serum-starved SH-SY5Y cells was assessed (Figure 4). The SH-SY5Y cells were treated with NE52-QQ57 (1 μM) at pH 7.4 and incubated for 24 h in serum-free culture media, to evaluate the effect of NE52-QQ57 on cell morphology and viability. However, no morphological alteration or cellular toxicity was observed in the control SH-SY5Y, GPR4-OE, or GPR4-KO cells (Figure 4A).

**Figure 4.** The effect of GPR4 antagonist, NE52-QQ57, on the cellular morphology and on GPR4 mRNA expression in MPP+-treated SH-SY5Y cells that were stably GPR4-OE or GPR4-KO. 24 h serum-starved SH-SY5Y cells were treated for 1 h with NE52-QQ57 followed by either a 24 h incubation or MPP<sup>+</sup> (1 mM) stimulation for 24 h in serum-free culture media. (**A**) The cell viability and morphology of SH-SY5Y, GPR4-OE, and GPR4-KO cells that were pre-treated for 1 h with NE52-QQ57. (**B**) An RT-PCR highlighting the mRNA expression of GPR4 and GAPDH in SH-SY5Y, GPR4-OE, and GPR4-KO cells pre-treated for 1 h with NE52-QQ57. (**C**) A semi-quantification of GPR4 mRNA expression 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.

For an RT-PCR, RNA was isolated from the cells pre-treated with NE52-QQ57 (1 μM) at pH 7.4; this was incubated for an additional 24 h in serum-free media, with or without MPP<sup>+</sup> (1 mM) stimulation. The resulting levels of GPR4 mRNA expression illustrated that the NE52-QQ5 (100 nM) had effectively blocked the expression of GPR4 on the control SH-SY5Y and the GPR4-OE cells, similar to genetically GPR4-KO cells (Figure 4B).

A semi-quantitative analysis (Figure 4C) of the RT-PCR bands demonstrated that the NE52-QQ5 significantly reduced GPR4 expression in the SH-SY5Y (0.02 ± 0.001 folds) and the GPR4-OE cells (0.29 ± 0.02 folds), compared with the nontreated SH-SY5Y and GPR4-OE cells, respectively. Moreover, treatment with NE52-QQ5 (100 nM) decreased GPR4 mRNA expression in the MPP<sup>+</sup> (1 mM) treated SH-SY5Y (0.9 ± 0.06 folds) and GPR4-OE cells (0.06 ± 0.004 folds), compared with the MPP+-stimulated SH-SY5Y and GPR4-OE cells, respectively.
