Hexachloronaphthalene Induces Mitochondrial-Dependent Neurotoxicity via a Mechanism of Enhanced Production of Reactive Oxygen Species

Figure 5

PCN67 induces intracellular calcium rise and plasma membrane depolarization. (a) Differentiated PC12 cells were treated with PCN67 for 72 h in the presence of intracellular calcium indicator—GcAMP3 and (b) propidium iodide (7.5 μM). The cells were placed into an environmental chamber with controlled temperature and CO₂ concentration, and the fluorescence changes were recorded every 4 h using an Axio Observer 7 Marianas™ Microscope equipped with 63x objective. The fluorescence of single cells was processed as ΔF/F₀ after background subtraction. (c) Representative images of cells stained with propidium iodide (7.5 μM) following treatment with PCN67 (IC₅₀) for 72 h in the presence or absence of calcium in the culture media. Scale bar 10 μm. (d) Chelation of intracellular calcium partially protected from cell death. BAPTA-AM (5 μM) was added to the culture 48 h after PCN67 treatment, and the viability was determined the day after. Vehicle-treated cells were taken as 100%. (e) Plasma membrane potential was measured 72 h following PCN67 treatment using DiSBAC₂ (1 μM). Cells were loaded with a dye for 30 min, and the fluorescence was analyzed using flow cytometry. and .

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