Fig. 5

Assessment of ROS accumulation, antioxidant gene expression, and mitochondrial network alterations after PCBs treatment in Dictyostelium. Ax4 cells were treated with 50 µM of PCB 138 or PCB 153 for 3 days. (A) ROS levels were indirectly measured by adding DHE to the cells and monitoring fluorescence over time (emission/excitation 560/590 nm, ± 20 nm). (B) Quantification of ROS production rates expressed as relative fluorescence units (RFU). Vehicle sample was used as control (= 1). (C) Expression levels of genes involved in oxidative stress response (sod1, sod2, sodA and sodB) were evaluated by qPCR. (D) Confocal imaging of mitochondrial morphology was performed on Ax4 cells expressing Fxn-GFP, and images were skeletonized for morphometric analysis (63x/1.40 oil objective; scale bar = 2 µm). The panel shown correspond to the original confocal images obtained. (E) ImageJ MiNa plugin was used to measure the ratio of mitochondrial area (footprint), branches length (µm) and network branches mean (mean number of connected lines for each structure). Three independent biological replicates were used for ROS measurement and qPCR data analysis. One-way ANOVA with Dunnett’s multiple comparisons test was used for statistical significance (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).