Fig 1: USP14 stabilizes MFG-E8 in BEAS-2B cells and HBE cells.A The indicated deubiquitinases were each transfected into BEAS-2B cells. Forty-eight hours later, cell lysates were subjected to western blot. B Increasing concentrations of USP14 plasmids were transfected into BEAS-2B cells and HBE cells. MFG-E8 protein expression was detected. C Empty vectors or USP14-overexpressed plasmids (2 μg/ml) were transfected into BEAS-2B cells and HBE cells. The cells were then treated with CHX for various time points prior to immunoblotting analyses. Quantification of MFG-E8 levels relative to GAPDH is shown in the right-hand panel. D Negative control siRNA or USP14 siRNA (50 nM) were transfected into BEAS-2B cells and HBE cells. The cells were then treated with CHX for various time points prior to immunoblotting analyses. Quantification of MFG-E8 levels relative to GAPDH was shown in the right-hand panel. Data are presented as the mean ± SD of three independent experiments.
Fig 2: MFG‐E8 gene knockout promotes CSE-induced ferroptosis in mouse lung tissues.A MFG‐E8 gene identification in C57BL/6 mice. B HE staining of lung slides. C Morphometric measurements of MLI (μm) and DI (%). WT-Ctrl: WT mice exposed to PBS. WT-CSE: WT mice exposed to CSE. KO-Ctrl: MFG-E8 KO mice exposed to PBS. KO-CSE: MFG-E8 KO mice exposed to CSE. D The morphological changes of mitochondria observed by transmission electron microscopy. E Ferric iron (brown) deposits stained with PERLS-DAB staining. F IHC staining of ferroptosis-related proteins (GPx4 and SLC7A11). G The average optical densities of GPx4 and SLC7A11 in IHC staining. H Western blot analyses of GPx4 and SLC7A11 protein levels. I Levels of GPx4 and SLC7A11 mRNA. Data are presented as the mean ± SD of three independent experiments. *P < 0.05, compared between the marked groups. **P < 0.01, compared between the marked groups. ***P < 0.001, compared between the marked groups.
Fig 3: MFG-E8 silencing induces ferroptosis in BEAS-2B cells and HBE cells.A The morphological changes of mitochondria observed by transmission electron microscopy. B Ferroptosis-related proteins (GPx4 and SLC7A11) were detected by western blot. C Levels of GPx4 and SLC7A11 mRNA. D Cell viability of MFG-E8 knockdown cells incubated with different concentrations of RSL3 for 24 h using CCK-8 assays. E Lipid peroxidation determined by C11-BODIPY staining. Data are presented as the mean ± SD of three independent experiments. *P < 0.05, compared between the marked groups. **P < 0.01, compared between the marked groups. ***P < 0.001, compared between the marked groups.
Fig 4: USP14/MFG-E8 axis involves in CSE-induced ferroptosis in BEAS-2B cells and HBE cells.A Cells were stimulated with CSE for 24 h and/ or MG132 for 5 h before collection prior to immunoblotting for MFG-E8, GPx4, and SLC7A11. B Empty vectors or USP14-overexpressed plasmids (2 μg/ml) were transfected into cells. Before the cells were collected, the transfected cells were stimulated with CSE for 24 h. USP14, MFG-E8, GPx4, and SLC7A11 were detected by western blot. C Negative control siRNA or USP14 siRNA (50 nM) were transfected into cells. Before the cells were collected, the transfected cells were treated with CSE for 24 h, or combined with MG132 for 5 h. USP14, MFG-E8, GPx4, and SLC7A11 were detected by western blot. D Negative control siRNA or USP14 siRNA were (50 nM) transfected into cells. Before the cells were collected, the transfected cells were treated with CSE for 24 h, or pretreated with rhMFG-E8 for 2 h followed by CSE treatment for 24 h. USP14, MFG-E8, GPx4, and SLC7A11 were detected by western blot. Data are presented as the mean ± SD of three independent experiments. *P < 0.05, compared between the marked groups. **P < 0.01, compared between the marked groups. ***P < 0.001, compared between the marked groups.
Fig 5: Cigarette smoke exposure diminishes MFG-E8 protein expression in vivo and in vitro.A IHC staining of MFG-E8 in lung tissues of COPD patients and healthy controls. The average optical density of MFG-E8 in human lung tissues is plotted in the right-hand panel. B IHC staining of MFG-E8 in lung tissues of mice. Control: WT mice exposed to PBS. CSE: WT mice exposed to CSE. The average optical density of MFG-E8 in mouse lung tissues is plotted in the right-hand panel. C Western blot analyses of MFG-E8 protein levels in lung tissues of COPD patients and healthy controls. D Western blot analyses of MFG-E8 protein levels in lung tissues of mice. Control: WT mice exposed to PBS. CSE: WT mice exposed to CSE. E Western blot analyses of MFG-E8 protein levels in BEAS-2B cells and HBE cells exposed to CSE. Data are presented as the mean ± SD. **P < 0.01, compared between the marked groups. ***P < 0.001, compared between the marked groups.
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