Fig. 5: DRP-1-dependent mitochondrial dysfunction caused by dysregulated fission in the artery of KD murine model.

A Representative immunofluorescence images for detecting NOX4 (green) expressions within aorta, DAPI (blue) (n = 5 per group). Scale bar: 50 μm. B NOX4 fluorescence intensity. C HUVECs were pre-treated with 10 μM of NOX4 inhibitor GKT137831 or Mas for 2 h, then LPS for 24 h. Dihydroethidium (DHE; 10 mM) was added to cells for another 30 min. Pictures were collected. Cells were observed at 100 × magnification. D Mitochondrial membrane potential was measured by using JC-10 dye. Graphical representation of the ratio of JC-1 aggregates to JC-10 monomers. Scale bars, 20 μm. E The appearance of mitochondria was observed by TEM. Mitochondrial fragmentation was shown in enlarge image in red frame. F Representative immunofluorescence images for DRP-1 (magenta) expressions of aortic endothelium, DAPI (blue) (n = 5 per group). Scale bar: 50 μm. G DRP-1 fluorescence intensity. H–J HUVECs were pre-treated with Mas (3 μM) for 2 h and then, LPS was applied to induce inflammatory injury together with Mas for another 48 h. Model group was treated with LPS only for 48 h. Representative western blot analysis to determine the proteins expression of DRP-1 and MFF in HUVEC cells. K Representative immunofluorescence images for double-labeling of NOX4 (red) and DRP-1 (yellow) expressions of aorta, DAPI (blue) (n = 3 per group). Scale bar: 50 μm. L DRP-1-dependent mitochondrial dysfunction was described in a graphical representation. One-way ANOVA was followed by post hoc Tukey’s test. *P < 0.05, vs vehicle group. ***P < 0.001 vs vehicle or Ctrl group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs model group.