Fig. 5.

TGF-β is involved in fibroblast activation by promoting the accumulation of iron in cells. (a–b) Intracellular Fe²⁺ levels in α-SMA⁺ cells in lung tissue of mice were assessed by flow cytometry using FeRhoNox-1 on Day 7 and Day 14 after bleomycin administration. Cells were first gated in α-SMA-FITC positive, and then subjected to analysis of Fe²⁺-PE fluorescent intensity. n = 3 mice per group. Statistical analysis was performed using the Student's t-test. ***P < 0.001. (c) Fluorescence microscopy images showing intracellular Fe²⁺ levels in MRC-5 stimulated with TGF-β, SB431542 (SB) and DFO using FeRhoNox-1. DAPI (blue). FeRhoNox-1 (Red). TGF-β: 10 ng/ml. SB: 1 μM. DFO: 50 μM. Scale bars, 200 μm n = 3 biological replicates. Statistical analysis was performed using one-way ANOVA followed by multiple comparisons. *P < 0.05, ***P < 0.001. (d) Fluorescence microscopy images showing intracellular Fe²⁺ levels in MRC-5 stimulated with FAC and DFO using FeRhoNox-1. DAPI (blue). FeRhoNox-1 (Red). FAC: 50 μM. DFO: 50 μM. Scale bars, 200 μm n = 3 biological replicates. Statistical analysis was performed using one-way ANOVA followed by multiple comparisons. ***P < 0.001. (e) Cell viability was determined by CCK8 after 72 h of treatment with FAC and DFO. FAC: 50 μM. DFO: 50 μM n = 4 biological replicates. Statistical analysis was performed using one-way ANOVA followed by multiple comparisons. *P < 0.05, **P < 0.01, ****P < 0.0001. (f) Cell viability was determined by CCK8 after 72 h of treatment with FAC and DFO. FAC: 50 μM. DFO: 50 μM n = 5 biological replicates. Statistical analysis was performed using one-way ANOVA followed by multiple comparisons. **P < 0.01, ****P < 0.0001. (g) Expression of α-SMA and COL1A1 in MRC-5 and IMR90 was assessed by Western blot after 72 h of treatment with the indicated doses of FAC and DFO. DFO: 50 μM. (h) Expression of α-SMA and COL1A1 in MRC-5 was assessed by Western blot after 72 h of treatment. TGF-β: 10 ng/ml. SB: 1 μM. DFO: 50 μM. (i) Confocal microscopy showing α-SMA in MRC-5 after 72 h of treatment with TGF-β, SB and DFO. DAPI (blue). α-SMA (red). TGF-β: 10 ng/ml. SB: 1 μM. DFO: 50 μM. Scale bars, 20 μm. (j) Representative microscopic images and the quantitation of MRC-5 cells that transferred through the transwell in the migration assay and invasion assays. Scale bars, 200 μm. TGF-β: 10 ng/ml. SB: 1 μM. DFO: 50 μM n = 6 visual fields. Statistical analysis was performed using one-way ANOVA followed by multiple comparisons. ****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.)