Figure 8.

Phosphatidylinositol biphosphate (PIP₂) calcium signalling and intracellular calcium levels in MPP⁺-treated SH-SY5Y cells that were stably GPR4-OE or GPR4-KO. 24 h serum-starved SH-SY5Y cells were treated with MPP⁺ (1 mM) for 24 h in serum-free culture media for the purpose of immunoblotting. The SH-SY5Y cells were then treated with H₂O₂ (200 µM) for 2 h 30 min in serum-free culture media and subjected to a Fluo-4 AM fluorescence assay and fluorescence microscopy. Detection of the Fluo-4 AM fluorescence intensity and related imaging were carried out according to the manufacturer’s instructions. Cells were counter-stained with Hoechst dye. (A) An immunoblot of the PIP₂ and β-Actin. (B) A quantitative analysis of the Fluo-4 AM fluorescence intensity in H₂O₂- (200 µM) treated cells. (C) Fluo-4 AM calcium imaging of the intracellular calcium level. 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.