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Figure 3: Mitochondrial ROS overproduction mediates DHA-induced apoptosis and autophagy. (a), (b) Increased levels of intracellular ROS induced by DHA in PC3 and DU145 cells. DHE-loaded PC3 and DU145 cells were examined for intracellular ROS accumulation by microscopy (a) and flow cytometry (b) after incubating with 50 μM DHA in the absence or presence of 5 mM NAC for 2 h (scale bar, 200 μm). (c), (d) Effect of the ROS scavenger NAC on the reduction in cell viability induced by DHA. PC3 (c) and DU145 (d) cells were exposed to 50 μM DHA and/or 5 mM NAC for 12 h and cell viability was measured by MTT. Data are displayed as means ± SD. ( ; ). (e) Effect of NAC on the levels of cleaved PARP and on the expression levels of LC3-II in DHA-treated cells. PC3 (left) and DU145 (right) cells were exposed to 50 μM DHA and/or 5 mM NAC for 12 h, and PARP cleavage and the expression levels of LC3-II were examined by Western blot analysis. Black triangle indicates the lipidated form LC3B (LC3-II), and asterisk indicates nonlipidated form of LC3B (LC3-I). (f) Increased levels of mitochondrial ROS induced by DHA. MitoSOX-loaded PC3 (top) and DU145 (bottom) cells were examined for mitochondrial ROS levels by flow cytometry after incubating with 50 μM DHA in the absence or presence of 5 mM NAC for 2 h.