Fig. 7.

Effects of Fundc1 regulation on NET formation and endothelial mitochondrial quality control. A. Intestinal CitH3-DNA and MPO-DNA complexes were assayed using ELISAs. B–C. Protein expression levels of Ly6G and CitH3 were measured by Western blot analysis. D-E. Intestinal CD31-positive endothelial cells were isolated with CD31-coated magnetic beads. Mitochondrial (MitoTracker) and lysosomal (LysoTracker) immunofluorescence colocalization was performed to explore the mitophagy level. F-G. Endothelial mitophagy parameters (p62, LC3 II/I ratio and mito-LC3 II) and a mitochondrial protein (Tim23) were examined using western blotting. The relative grayscale values were measured using ImageJ. H–I. Cytc levels in the cytosol and isolated mitochondria were examined by western blotting. J-K. Quantification of cytoplasmic and mitochondrial ROS in the intestinal endothelium was conducted using DCFH‐DA and MitoSOX Red probes. L. Mitochondrial membrane potential changes in endothelial cells were detected by JC-1 staining. M-N. Key proteins of mitochondrial fusion (Mfn1 and Opa1) and fission (Mff and Drp1) were examined by a Western blot assay. O–P. Immunofluorescence confocal imaging of mitochondrial (Tom20, green) and Drp1 (red) colocalization was used to evaluate mitochondrial fission. AAV, adeno-associated virus; NC, negative control. Data are shown as the means ± SD, ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.