Journal: Journal of Advanced Research

Article Title: NCOA4-Mediated Ferritinophagy Induces Ferroptosis and Enriches Ferritin-Containing EVs via Ferritin Phase Separation to Promote Mechanical Ventilation-Induced Pulmonary Fibrosis

doi: 10.1016/j.jare.2025.07.043

Figure Lengend Snippet: Ferritinophagy aggravated MV-induced pulmonary fibrosis through regulating ferroptosis. (A, B). Immunoblot and quantitative analysis showing the expression levels of ferritinophagy markers NCOA4 and FTH in MLE-12 cells with and without MS. n = 3 per group. (C, D). Immunoblot and quantitative analysis showing the expression levels of NCOA4 and FTH in the lung homogenates. n = 4–6 per group. (E, F). Immunoblot and quantitative analysis showing the expression levels of SLC7A11 and GPX4 in the lung homogenates. n = 4–6 per group. (G, H). Immunoblot and quantitative analysis showing the expression levels of SLC7A11, GPX4 and FTH in DFO-treated MLE-12 cells with and without MS. n = 3 per group. Representative immunofluorescence staining images and relevant quantitative analysis for the epithelial marker E-cadherin (Green) and NCOA4 (Red) (I), or FTH (Green) and NCOA4 (Red) in the lung tissues (J). White arrows point to the colocalization of the two markers. Nuclei was stained by DAPI (blue). Scale bars correspond to 20 μm. MFI, mean fluorescence intensity. (K, L). Immunoblot and quantitative analysis showing the expression levels of fibronectin and α-SMA in the lung homogenates. n = 4–6 per group. (M, N). Representative histopathologic images of H&E and Masson’s staining comparing the lung injury and collagen deposition. Original magnification x 200. Scale bars correspond to 100 μm. All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The slides were then washed and mounted using DAPI Fluoromount-G (0100–20, Southern Biotechnology, USA).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Marker, Fluorescence

Journal: Journal of Advanced Research

Article Title: NCOA4-Mediated Ferritinophagy Induces Ferroptosis and Enriches Ferritin-Containing EVs via Ferritin Phase Separation to Promote Mechanical Ventilation-Induced Pulmonary Fibrosis

doi: 10.1016/j.jare.2025.07.043

Figure Lengend Snippet: Activation of ANG II/AGTR1 pathway initiated NCOA4-mediated ferritinophagy to aggravate MV-induced pulmonary fibrosis. (A, B). Immunoblot and quantitative analysis showing the expression levels of ANG II and AGTR1 in the lung homogenates. n = 4–6 per group. (C, D). Immunoblot and quantitative analysis showing the expression levels of NCOA4 and FTH in the lung homogenates. n = 4–6 per group. (E, F). Immunoblot and quantitative analysis showing the expression levels of SLC7A11 and GPX4 in the lung homogenates. n = 4–6 per group. Representative immunofluorescence staining images and relevant quantitative analysis for the epithelial marker E-cadherin (Green) and NCOA4 (Red) (G), or FTH (Green) and NCOA4 (Red) in the lung tissues (H). White arrows point to the colocalization of the two markers. Nuclei was stained by DAPI (blue). Scale bars correspond to 20 μm. MFI, mean fluorescence intensity. (I, J) Immunoblot and quantitative analysis showing the expression levels of fibronectin and α-SMA in the lung homogenates. n = 4–6 per group. (K, L). Representative histopathologic images of H&E and Masson’s staining comparing the lung injury and collagen deposition. Original magnification x 200. Scale bars correspond to 100 μm. All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The slides were then washed and mounted using DAPI Fluoromount-G (0100–20, Southern Biotechnology, USA).

Techniques: Activation Assay, Western Blot, Expressing, Immunofluorescence, Staining, Marker, Fluorescence

Journal: Journal of Advanced Research

Article Title: NCOA4-Mediated Ferritinophagy Induces Ferroptosis and Enriches Ferritin-Containing EVs via Ferritin Phase Separation to Promote Mechanical Ventilation-Induced Pulmonary Fibrosis

doi: 10.1016/j.jare.2025.07.043

Figure Lengend Snippet: Suppression of NCOA4 inhibited ferroptosis and alleviated MV-induced pulmonary fibrosis. (A, B). Immunoblot and quantitative analysis showing the expression levels of NCOA4 and FTH in the lung homogenates. n = 4–6 per group. (C, D). Immunoblot and quantitative analysis showing the expression levels of SLC7A11 and GPX4 in the lung homogenates. n = 4–6 per group. Representative immunofluorescence staining images and relevant quantitative analysis for the epithelial marker E-cadherin (Green) and NCOA4 (Red) (E), or FTH (Green) and NCOA4 (Red) (F) in the lung tissues. White arrows point to the colocalization of the two markers. Nuclei was stained by DAPI (blue). Scale bars correspond to 20 μm. MFI, mean fluorescence intensity. (G, H). Immunoblot and quantitative analysis showing the expression levels of fibronectin and α-SMA in the lung homogenates. n = 4–6 per group. (I, J). Representative histopathologic images of H&E and Masson’s staining comparing the lung injury and collagen deposition. Original magnification x 200. Scale bars correspond to 100 μm. All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The slides were then washed and mounted using DAPI Fluoromount-G (0100–20, Southern Biotechnology, USA).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Marker, Fluorescence

Journal: Journal of Advanced Research

Article Title: NCOA4-Mediated Ferritinophagy Induces Ferroptosis and Enriches Ferritin-Containing EVs via Ferritin Phase Separation to Promote Mechanical Ventilation-Induced Pulmonary Fibrosis

doi: 10.1016/j.jare.2025.07.043

Figure Lengend Snippet: Mechanical ventilation enriched EVs to promote activation of lung fibroblast through iron overload. (A). GO enrichment analysis of differential expressed genes in the type II alveolar epithelial cells and fibroblasts originated from the MV group versus the control group. (B). Immunoblot analysis of the characteristic markers, Alix and CD63, and ferritin of EVs from control-BALF and MV-BALF. n = 3 per group. (C). NTA analysis of the concentration and size distribution of EVs from control-BALF and MV-BALF. (D). TEM images of EVs from control-BALF and MV-BALF. Original magnification x 80,000. Scale bars correspond to 200 nm. (E). Heatmap of the correlation strength of cellular interactions between different cells. (F). Cellchat analysis showing the intracellular interaction strength between AT2 or fibroblasts and other cell types in the MV group. (G, H). Immunoblot and quantitative analysis of fibronectin, α-SMA and FTH in MRC-5 cells treated with EVs. n = 3 per group. (I). Representative images of ferrous iron (red) in the MRC-5 cells treated with PKH-67 labelled EVs (green) by the confocal microscopy. Nuclei was stained by DAPI (blue). Scale bars correspond to 10 μm, and n = 3 biologically independent samples. All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The slides were then washed and mounted using DAPI Fluoromount-G (0100–20, Southern Biotechnology, USA).

Techniques: Activation Assay, Control, Western Blot, Concentration Assay, Confocal Microscopy, Staining

Journal: Journal of Advanced Research

Article Title: NCOA4-Mediated Ferritinophagy Induces Ferroptosis and Enriches Ferritin-Containing EVs via Ferritin Phase Separation to Promote Mechanical Ventilation-Induced Pulmonary Fibrosis

doi: 10.1016/j.jare.2025.07.043

Figure Lengend Snippet: MV induced ferritinophagy promoted the transfer of iron from EVs to activate lung fibroblast. (A, B). Immunoblot and quantitative analysis of fibronectin, α-SMA and FTH in MRC-5 cells treated with BALF-EVs generated from chloroquine intervened mice. n = 3 per group. (C). Representative images of ferrous iron (red) in the MRC-5 cells treated with PKH-67 labelled EVs (green) by the confocal microscopy. Nuclei was stained by DAPI (blue). (D). Immunofluorescence of α-SMA (red) in MRC-5 cells treated with BALF-EVs generated from chloroquine intervened mice. Nuclei was stained by DAPI (blue). (E, F). Immunoblot and quantitative analysis of fibronectin, α-SMA and FTH in MRC-5 cells treated with BALF-EVs generated from NCOA4-knockdown mice administrated by AAV. n = 3 per group. (G). Representative images of ferrous iron (red) in the MRC-5 cells treated with PKH-67 labelled EVs (green) by the confocal microscopy. Nuclei was stained by DAPI (blue). (H). Immunofluorescence of α-SMA (red) in MRC-5 cells treated with BALF-EVs generated from NCOA4-knockdown mice administrated by AAV. Nuclei was stained by DAPI (blue). Scale bars correspond to 10 μm, and n = 3 biologically independent samples. All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The slides were then washed and mounted using DAPI Fluoromount-G (0100–20, Southern Biotechnology, USA).

Techniques: Western Blot, Generated, Confocal Microscopy, Staining, Immunofluorescence, Knockdown