*4.8. Assessment of Mitochondrial Membrane Potential (MMP)*

A JC-10-based Mitochondrial Membrane Potential Assay Kit (Abcam; Cambridge, United Kingdom) was employed to assess MMP, while a fluorescence microscope was utilised to visualise the JC-10 staining, according to the manufacturer's instructions. In brief, SH-SY5Y cells (2.2 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/mL) were cultured in black, 96-well plates for quantification and in 6-well plates for imaging in DMEM/F12 without phenol red. Then, 60–70% confluence cells were stimulated with MPP<sup>+</sup> (1 mM) for 24 h in serum-free media. The cells were incubated with a JC-10 dye loading solution at 37 ◦C for 1 h and protected from the light. For the 96-well plates, their fluorescence intensities (λex = 490/λem = 525 nm) and (λex = 540/λem = 590 nm) the red-green fluorescence ratios were measured using a fluorescence microplate reader (Molecular Device; Sunnyvale, CA, USA), while confocal images were acquired with a Nikon Eclipse Ts2-FL diascopic and epi-fluorescence illumination microscope.

#### *4.9. Detection of Intracellular Calcium*

Intracellular calcium was assessed with a Fluo-4 AM dye (Abcam; Cambridge, United Kingdom) and confocal microscopy, following the manufacturer's instructions. Fluo-4 AM was diluted in DMSO containing 2 mM probenecid and 0.02% pluronic F-127. In brief, the SH-SY5Y cells (2.2 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/mL) were cultured in black, 96-well plates for quantification and in 6-well plates for imaging in DMEM/F12 without phenol red. Then, 60–70% confluence cells were stimulated with H2O2 (200 μM) for 2 h 30 min in serum-free media. The cells were washed with PBS containing probenecid (2 mM) at room temperature. The cells were incubated with the Fluo-4 AM (2 μM) dye loading solution at 37 ◦C for 30 min and protected from light, then washed with PBS containing probenecid (2 mM) at room temperature for 30 min. For the 96-well plates, the fluorescence intensities (λex = 488/λem = 515 nm) were measured using a fluorescence microplate reader (Molecular Device; Sunnyvale, CA, USA), and confocal images were acquired with a Nikon Eclipse Ts2-FL diascopic and epi-fluorescence illumination microscope.
