Fig. 5: Macrophage-derived exosomal APN/CD13 promotes lung epithelial cell necroptosis by binding to the receptor TLR4. | Communications Biology

Fig. 5: Macrophage-derived exosomal APN/CD13 promotes lung epithelial cell necroptosis by binding to the receptor TLR4.

From: Macrophage-derived exosomal aminopeptidase N aggravates sepsis-induced acute lung injury by regulating necroptosis of lung epithelial cell

Fig. 5

a The APN expression levels were measured using western blotting and qRT-PCR in macrophages treated with LV-shNC or LV-APN. b Isolated exosomes under the electron microscope. Scale bar: 100 nm. c ELISA detection of APN expression in exosomes derived from macrophages transfected with shNC or shAPN in the presence of LPS. Exosomes from PBS-treated macrophages were used as the negative control. Ctrl-exo, exosomes from macrophages without LPS, LPS-exoshNC exosomes from shNC macrophages with LPS treatment, LPS -exoshAPN exosomes from shAPN macrophages with LPS treatment. After lung epithelial cells were cocultured with these exosomes for 24 h in the presence of LPS (1 µg/mL), the expression of APN was measured by western blotting (d), the viability of the cells was examined using the CCK8 assay (e), analyses of Annexin V/PI staining using flow cytometry (f). g The characteristic morphological changes in necroptosis in LPS-exoshNC-treated BEAS-2B cells were observed using TEM. h Western blotting analysis of the expression of apoptosis-related protein cleaved caspase-8 and cleaved caspase-3 in BEAS-2B cells. i Measurement of intracellular ROS level via flow cytometry, (j) the mitochondrial structures were observed using an electron microscope. k RT-PCR analysis of the mRNA expression of IL-6, TNF-α and IL-1β. l The expression of p-p65, RIPK1, RIPK3, and p-MLKL was detected using western blotting. m Colocalization of APN (green) and TLR4 (red) in LPS-exoshNC-treated BEAS-2B cells using confocal microscopy. Scale bar, 50 μm. n Cell lysates from BEAS-2B cells precipitated with agarose beads were pulled down and probed for TLR4 and APN protein levels with control IgG or TLR4 mAb. o PLA was performed using specific antibodies against APN and TLR4. Nuclei were stained with DAPI. Scale bars represent 15 μm. PLA signals per cell were quantified by Image software-assisted analysis. All results are based on three measurements. The information is presented as the mean ± SD (n = 3), *P < 0.05, **P < 0.01.

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