Fig 1: NEDD4L stimulates ß-catenin ubiquitination, represses CTHRC1/HIF-1a axis, and alleviates IPF in vivo. A: qRT-PCR to measure the expression of NEDD4L in the lung tissues of mice treated with PR-619 and sh-NEDD4L; B: Western blot to measure the protein level of ß-catenin, CTHRC1 and HIF-1a in the lung tissues of mice treated with PR-619 and sh-NEDD4L; C: Representative images and scores of the pathological changes in the lung tissues of mice treated with PR-619 and sh-NEDD4L (n = 6); D: Masson staining to examine lung tissue fibrosis in mice treated with PR-619 and sh-NEDD4L (n = 6); E: Detection of hydroxyproline content in the lung tissues of mice treated with PR-619 and sh-NEDD4L (n = 6); F: Resistance and compliance plethysmograph to detect lung functions of mice treated with PR-619 and sh-NEDD4L (n = 6). * p <0.05, ** p < 0.01, *** p < 0.001 versus the IPF + DMSO group. Measurement data were summarized as mean ± standard deviation. Independent sample t test was applied for comparison between data of two groups; one-way ANOVA was adopted for comparison among data of multiple groups. The experiment was performed in triplicates.
Fig 2: NEDD4L stimulates β-catenin ubiquitination and thereby restricts the activation of the CTHRC1/HIF-1α axis. A: E3 ubiquitin ligase of β-catenin (CTNNB1) predicted by UbiBrowser database; B: Immunoprecipitation to determine β-catenin ubiquitination in LFs; C: Immunohistochemistry to detect β-catenin expression in 35 IPF and 28 normal lung tissues (scale bar = 50 μm); D: Pearson correlation analysis on β-catenin and CTHRC1 expression in 35 cases of IPF lung tissues; E: ChIP to evaluate β-catenin enrichment in the promoter region of CTHRC1 gene; F: Western blot to determine levels of β-catenin and CTHRC1 proteins in LFs of each group; G: Western blot to determine the protein levels of β-catenin, CTHRC1 and HIF-1α in LFs of each group. * p <0.05, ** p < 0.01, *** p < 0.001 versus the Normal, IgG, DMSO, or si-NC group. Measurement data were summarized as mean ± standard deviation. Independent sample t test was applied for comparison between data of two groups; one-way ANOVA was adopted for comparison among data of multiple groups. The experiment was performed in triplicates.
Fig 3: NEDD4L regulates the CTHRC1/HIF-1a axis to repress the proliferative, invasive and differentiative abilities of LFs. A: Negative correlation between NEDD4L and CTHRC1 expression in GSE10667 microarray; B: Pearson correlation analysis on NEDD4L and CTHRC1 expression in lung tissues of IPF patients (n = 35); C: Western blot to measure the protein levels of CTHRC1 and HIF-1a in LFs; D: qRT-PCR to determine the expression of CTHRC1 and HIF-1a in LFs of each group; F: CCK-8 assay to detect the viability of LFs; G: Transwell to detect the invasive ability of LFs; H: Immunofluorescence to measure the level of a-SMA in cells of each group; I: Western blot to determine the levels of fibrosis-related proteins in LFs of each group. * p <0.05, ** p < 0.01, *** p < 0.001 versus the Mock or oe-NC group. Measurement data were summarized as mean ± standard deviation. Independent sample t test was applied for comparison between data of two groups; one-way ANOVA was adopted for comparison among data of multiple groups. The experiment was performed in triplicates and in the presence of TGF-ß1.
Fig 4: The mechanism graph of the regulatory network and function of NEDD4L in pulmonary fibrosis. NEDD4L stimulates β-catenin ubiquitination, represses the CTHRC1/HIF-1α axis, attenuates the biological potential of lung fibroblasts, and thus alleviates pulmonary fibrosis.
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