Fig. 6.

TGF-β enhances TFRC expression in fibroblasts by promoting the formation and nuclear localization of TAZ/TEAD4 complex. (a) Expression of α-SMA, COL1A1, TFRC, FTH and SLC40A1 in MRC-5 was assessed by Western blot after 72 h of treatment with the indicated doses of TGF-β. (b) Expression of TFRC in MRC-5 and PLFs were assessed by qPCR after 48 h of treatment with the indicated doses of TGF-β. n = 4 biological replicates. 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) MFI of TFRC-PE in α-SMA⁺ cells in lung tissue of mice was assessed by flow cytometry on Day 14 after bleomycin administration. Left, representative flow cytometry result for MFI of TFRC-PE in α-SMA⁺ cells. Cells were first gated in α-SMA-FITC positive, and then subjected to analysis of TFRC-PE fluorescent intensity. Right, scatter plot showing the results for independent samples examined. n = 3 mice per group. Statistical analysis was performed using the Student's t-test. **P < 0.01. (d) Immunofluorescence showing TFRC and α-SMA in lung tissue of mice on Day 14 after bleomycin administration. DAPI (blue). TFRC (Red). α-SMA (Green). Scale bars, 10 μm. (e) Expression of α-SMA and TFRC in MRC-5 after TFRC knockdown with siRNA was assessed by Western blot after 72 h of treatment with TGF-β. TGF-β: 10 ng/ml. (f) Fluorescence microscopy images showing intracellular Fe²⁺ levels in MRC-5 after TFRC knockdown with siRNA using FeRhoNox-1 after 24 h of treatment with FAC. DAPI (blue). FeRhoNox-1 (Red). FAC: 2 μM. Scale bars, 200 μm. (g) Expression of α-SMA and TFRC in MRC-5 overexpressing TFRC was assessed by Western blot after 72 h of treatment with the indicated doses of FAC. (h) Fluorescence microscopy images showing intracellular Fe²⁺ levels in MRC-5 overexpressing TFRC using FeRhoNox-1 after 24 h of treatment with FAC. FeRhoNox-1 (Red). FAC: 2 μM. Scale bars, 200 μm. (i) Expression of TAZ in MRC-5 and PLFs was assessed by Western blot after 72 h of treatment with the indicated doses of TGF-β. (j) Expression of TAZ, TFRC and α-SMA in MRC-5 and PLFs was assessed by Western blot after 72 h of treatment with TGF-β and SB. TGF-β: 10 ng/ml. SB: 1 μM. (k) Confocal microscopy showing the expression and localization of TAZ and α-SMA in MRC-5 and PLFs after 48 h of treatment with TGF-β. DAPI (blue). TAZ (green). α-SMA (red). TGF-β: 10 ng/ml. SB: 1 μM. Scale bars, 50 μm. (l) TFRC promoter luciferase activity was determined in 293T cells transfected with pCMV6-Entry-TEAD4 plasmid using dual luciferase reporter assay. n = 4 biological replicates. 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.)