Taken together, our data suggested that FCCP as an O(2)(*-) generator, induces apoptosis via the depletion of intracellular GSH contents in Calu-6 cells. In contrast, BSO, a well-known inhibitor of GSH synthesis, aggravated GSH depletion, oxidative stress of O(2)(*-) and cell death in FCCP-treated cells. Treatment with thiol antioxidants, NAC and DTT, showed the recovery of GSH depletion and the reduction of O(2)(*-) levels in FCCP-treated cells, which were accompanied by the inhibition of apoptosis. Dashed box, cell populations are viable cells, as they are Annexin V and PI negative. Stable SOD mimetics, Tempol and Tiron did not change the levels of intracellular O(2)(*-), apoptosis and the loss of mitochondrial membrane potential (DeltaPsi(m)). U251 MG cells were treated with the uncoupler CCCP (5 mol/L) during glucose withdrawal and analyzed for viability using Annexin V/PI staining and flow cytometry 24 hours later. A depletion of intracellular GSH content was also observed after exposing cells to FCCP. Levels of intracellular O(2)(*-) were markedly increased depending on the concentrations (5-100 microM) of FCCP. Here, we investigated an involvement of O(2)(*-) and GSH in FCCP-induced Calu-6 cell death and examined whether ROS scavengers rescue cells from FCCP-induced cell death. In conclusion, our study suggests that the mitochondrial molecular landscape of Substantia nigra specimens of PD patients can be mirrored by the impaired dopamine homeostasis cellular model, thus supporting the hypothesis that alterations in this process could be a crucial pathogenetic event in PD.Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. Eventually, in transmission electron microscopy images, small electron dense deposits were observed in mitochondria of PD subjects, which are uniquely reproduced in dopamine-treated cells. Moreover, the accumulation of PTEN-induced putative kinase 1 (PINK1), a mitophagy marker, was not observed in both PD patients samples and cellular models. In this case, dopamine treatment better recapitulated the molecular picture of patients' samples. Opposite changes in the levels of two mitochondrial protein markers (voltage-dependent anion channels (VDACs) and cytochrome c oxidase subunit 5β (COX5β)) were observed. However, the effects of dopamine and MPP + treatments resulted to be different in terms of the mitochondrial damage induced. The cells were subsequently stained with MitoTracker Green and LysoTracker Red. As a result, we found that fusion impairment of the inner mitochondrial membrane is a common feature of both PD human samples and cellular models. Figure 8 Effect of CCCP-mediated mitophagy following CVB3 infection in H9C2 cardiomyocytes (A) H9C2 cells were treated with 25 M CCCP for 2 hours or infected with mock or CVB3 for 24 h (MOI 50). Indeed, SH-SY5Y cells were treated with either dopamine or 1-methyl-4-phenylpyridinium (MPP +) in order to highlight the effect of altered dopamine homeostasis and of complex I inhibition, respectively. In this work, we wanted to verify the molecular basis of altered mitochondrial dynamics and disposal in Substantia nigra specimens of sporadic PD patients, by the comparison with two cellular models of PD. Mitochondrial impairment is one of the most important hallmarks of Parkinson's disease (PD) pathogenesis. Image analysis methods and apparatus are used for distinguishing live and dead cells.The methods may involve segmenting an image to identify the region(s) occupied by one or more cells and determining the presence of a particular live-dead indicator feature within the region(s).In certain embodiments, the indicator feature is a cytoskeletal component such as tubulin.