*2.6. Knockout of GPR4 Decreases the Caspase-3 Activity and Lowers the ROS Generation in Neurotoxin-Stimulated SH-SY5Y Cells*

Caspases are important factors that trigger apoptosis. Caspase-3, in particular, is a crucial biomarker and executor of neuronal apoptosis [31]. To evaluate Caspase-3 activity, immunoblotting and a caspase activity assay were performed (Figure 6). SH-SY5Y cells were treated with MPP<sup>+</sup> (1 mM) for 24 h in serum-free media, while cell lysates were analysed through a western blot and a caspase activity assay. MPP+-treated GPR4-OE cells demonstrated a significant increase in their cleaved Caspase-3 protein levels, whereas knockout of GPR4 prevented an MPP<sup>+</sup> stimulated increase in the level of cleaved Caspase-3 (Figure 6A).

**Figure 6.** The Caspase-3 activity and intracellular ROS generation in MPP<sup>+</sup>- and H2O2-treated SH-SY5Y cells that were stably GPR4-OE or 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 for an immunoblot and a caspase activity assay. SH-SY5Y, GPR4-OE, and GPR4-KO cells were treated with H2O2 (300 μM) for 1 h in serum-free culture media for a 2 ,7 -dichlorofluorescein diacetate (DCFDA) assay. (**A**) An immunoblot of the pro caspase & cleaved Caspase-3 and β-Actin. (**B**) Caspase-3 activity was measured using a colorimetric assay kit (Sigma, CAS No. CASP-3-C) in MPP+-induced apoptotic cells. (**C**) The relative intracellular ROS level after 1 h of H2O2 (300 μM) treatment of 24 h serum-starved SH-SY5Y, GPR4-OE, and 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.

The Caspase-3 activity assay results were in complete agreement with the Caspase-3 activity demonstrated in the immunoblot data (Figure 6B). MPP+-treated GPR4-KO cells presented a lower level of caspase activity (1.55 <sup>±</sup> 0.03 folds) than the MPP+-treated SHSY-5Y (2.06 <sup>±</sup> 0.04 folds) and GPR4-OE cells (2.47 ± 0.011 folds; Figure 6B).

We evaluated the effects of GPR4 overexpression and knockout on H2O2-induced intracellular ROS generation in SH-SY5Y cells [19]. In our study, the protective effect of the knockout of GPR4 against H2O2 resulted in lower intracellular ROS levels measured in the SH-SY5Y cells. DCFDA, a fluorescent dye that in the presence of ROS is oxidised to fluorescent DCF, was utilised for the detection of intracellular ROS levels. Treatment of the SH-SY5Y cells with H2O2 (300 μM) for 1 h led to a marked increase in their intracellular ROS levels. In H2O2-treated GPR4-OE cells, the level of intracellular ROS generation was 1.56 ± 0.01 folds higher than that of the non-treated GPR4-OE cells, whereas the H2O2-treated GPR4-KO cells demonstrated a lower level of ROS generation (1.20 ± 0.04 folds) in comparison with both the H2O2-treated control SH-SY5Y (1.42 ± 0.001 folds) and the H2O2-treated GPR4-OE cells (1.56 ± 0.01 folds; Figure 6B).
