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. 2019 May 15;11(5):273. doi: 10.3390/toxins11050273

Figure 3.

Figure 3

Effect of EEP on reactive oxygen species (ROS) production and caspase-3/7 activity in copper-induced oxidative stress in P19 neuronal cells. At 24 h from the beginning of exposure to EEP (1–5 µg/mL) and/or CuSO4 (0.5 and 0.75 mM), accumulation of ROS was quantified by an assay based on the generation of fluorescent products from the 2′,7′- dichlorofluorescin diacetate. In physiological conditions small concentrations of EEP did not affect intracellular ROS amounts (a), whereas in the presence of 0.5 and 0.75 mM CuSO4 (b,c, respectively), EEP stimulated production of dangerous oxidative species. EEP applied alone also did not modify basal level of caspase-3/7 activity (d), but induced caspase activation in the presence of copper ions (e,f). Data are expressed as mean ± SEM from 4–6 independent experiments performed in triplicate. Data were analyzed by one-way ANOVA followed by Dunnett’s post hoc test after exposure to 0.5 mM CuSO4 and by t-test after exposure to 0.75 mM CuSO4 (a P < 0.05, b P < 0.01, c P < 0.001 and d P < 0.0001 versus copper-treated groups).