Fig. 4

C-Mito^HF induced oxidative stress, cytosolic free dsDNA release, subsequent cGAS activation and inflammatory response in brain ECs. A DCFH-DA probe and Mito-SOX probe were used to detect ROS level in bEnd.3 cells. Scale bar = 5 μm. B Analysis showed that C-Mito^HF significantly increased intracellular ROS level compared with C-Mito^Ctrl in bEnd.3 cells. n = 6, t test. C Analysis showed that C-Mito^HF significantly increased mitochondria-derived ROS level compared with C-Mito^Ctrl in bEnd.3 cells. n = 6, t test. D Cytosolic free dsDNA was detected by double-immunofluorescent staining with dsDNA antibody and TOM20 antibody. Scale bar = 5 μm. E Cytosolic free dsDNA level was evaluated. F cGAS expression in C-Mito^HF-treated bEnd.3 cells in vitro was evaluated by immunofluorescent staining. Scale bar = 5 μm. G Relative fluorescent intensity of cGAS in C-Mito^HF-treated bEnd.3 cells was analyzed. H cGAMP level in C-Mito^HF-treated bEnd.3 cells was measured. I Pro-inflammatory cytokines (TNF-α and IL-1β) production were measured in C-Mito^HF-treated bEnd.3 cells. n = 6, P < 0.05, ANOVA LSD test