Fig. 4

AA supplementation decreased intracellular and mitochondrial ROS (mt ROS) production and improved mitochondrial function. The effect of AA on intracellular ROS and mitochondrial ROS generation were respectively detected using DCFH-DA and Mito Sox fluorescent staining. JC-1 staining was used to determine the effects of AA on mitochondrial membrane potential stability. To further detect the effect of AA on mitochondrial function, intracellular ATP levels of each group were measured using commercially available ATP assay kit. A Representative images of DCFH-DA fluorescent staining. Green-stained DCFH-DA positive cells represent intracellular ROS and blue represents nuclei. The scale bar is 50 μm. B Quantification of DCFH-DA fluorescence intensity. C Representative images of Mito Sox confocal images. Red-stained Mito Sox-positive dots represent mtROS and blue represents nuclei. The scale bar is 30 μm. D Quantification of Mito Sox fluorescence intensities. E Representative images of JC-1 staining; the red images on the left represent JC-1 aggregates, and the green images represent JC-1 monomer. The blue images represent DAPI, and the right panel shows the merged images. F Quantification of JC-1 fluorescence intensities. G Quantification of ATP level. These data are expressed as the mean ± SD of 3 independent experiments. ***P < 0.001. Statistical analysis was carried out by a one-way ANOVA analysis. AA: arachidonic acid; a.u.: arbitrary units; DAPI: 4′,6-diamidino-2-phenylindole; DCFH-DA: 2,7- dichlorofluorescein diacetate; JC-1: 5,5′,6,6′-tetrachloro- 1,1′,3,3′-tetraethyl benzimidazolylcarbcyanine iodide ROS: reactive oxygen species