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. 2022 Oct 18;57:102509. doi: 10.1016/j.redox.2022.102509

Fig. 1.

Fig. 1

The accumulation of lipid peroxides and iron overload in lungs from bleomycin-induced pulmonary fibrosis mice. (a) Upper, lungs of mice from control (Ctrl) group and bleomycin (BLM) group on Day 21 after bleomycin administration. Bottom, representative images of H&E staining, Masson staining, and IHC staining for α-SMA, FAPα and TGF-β of lung tissue from control group and bleomycin-treated group at different days. Scale bars, 100 μm. (b) Scatter plot showing the percentage of the positive area of Masson staining and IHC staining in lung tissue. n = 3–4 mice per group. Statistical analysis was performed using one-way ANOVA followed by multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (c) Left, representative images of multicolor immunofluorescence for DAPI (blue), α-SMA (green) and TGF-β (orange) in lung tissue of mice on Day 14 after bleomycin administration. Scale bars, 50 μm. Right, scatter plot showing the percentage of α-SMA+ and TGF-β+ cells, mean fluorescence intensity (MFI) of α-SMA and the positive area of TGF-β in lung tissue of mice on Day 14 after bleomycin administration. Each dot represents a microscopic image, n = 15 images per group from 3 mice. Statistical analysis was performed using the Student's t-test. *P < 0.05, ****P < 0.0001. (d) Left, immunoblotting analysis of 4-HNE in lung tissues of mice on Day 7 and Day 14 after bleomycin administration. Right, quantitative analysis of 4-HNE from three mice. Statistical analysis was performed using the Student's t-test. *P < 0.05, **P < 0.01. (e) Intracellular Fe2+ levels in lung tissue of mice were assessed by flow cytometry using FeRhoNox-1 on Day 7 and Day 14 after bleomycin administration. Upper, representative flow cytometry result for Fe2+. Bottom, scatter plot showing the results from three mice. Statistical analysis was performed using one-way ANOVA followed by multiple comparisons. **P < 0.01, ****P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)