Fig. 5.
NET formation negatively impacted mitochondrial quality control in the microvascular endothelium of the intestine. A-B. Western blotting to assess mitophagy flux (p62, LC3 II/I ratio and mito-LC3 II) and mitochondrial protein (Tom20 and Tim23) levels in the intestinal endothelium of WT and Pad4ΔPMN mice. C-D. To evaluate the level of mitophagy, colocalization of mitochondria (MitoTracker) and lysosomes (LysoTracker) was analyzed using immunofluorescence. E-F. Western blotting was used to investigate the Cytc levels in both the cytosol and isolated mitochondria of intestinal endothelial cells. G. ATP generation was measured using an ATP assay kit. H–I. Cytoplasmic and mitochondrial ROS were measured using DCFH‐DA and MitoSOX Red probes. J. The mitochondrial membrane potential of isolated intestinal endothelial cells was examined by JC-1 staining. K-L. Western blotting was used to analyze the regulatory factors related to mitochondrial fusion (Mfn1 and Opa1) and fission (Mff and Drp1). M-N. Immunofluorescence detection of mitochondrial fission was accomplished by costaining Drp1 and mitochondria. Drp1 exhibits a propensity to interact with fragmented mitochondria. O–P. Morphological alterations in mitochondria were observed using immunofluorescence (Tom20) and TEM. The average mitochondrial length and the numbers of mitophagosomes were determined. TEM, transmission electron microscopy. Data are expressed as the means ± SD, ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.