SAHA inhibits lung fibroblast activation by increasing p66Shc expression epigenetically

Figures

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  FIGURE 1. SAHA inhibited TGF‐β1‐induced activation of lung fibroblasts. (A) The viability of MRC‐5 cells incubated with different concentrations of SAHA for 24 h (n = 3). (B, C) Western blot analysis and quantification of collagen I and α‐SMA protein levels in MRC‐5 cells treated with different concentrations of SAHA for 24 h in the presence of TGF‐β1 (n = 3). (D, E, and F) Immunofluorescence images and quantification of collagen I (green) and α‐SMA (red) in TGF‐β1‐induced MRC‐5 cells treated with different concentrations of SAHA (n = 3). The bars represent the mean ± SD of three separate experiments. * indicates p < 0.050, ** indicates p < 0.010, *** indicates p < 0.001, **** indicates p < 0.0001.

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  FIGURE 2. SAHA inhibits TGF‐β1‐induced proliferation and migration of MRC‐5 cells. (A) The CCK‐8 assay of MRC‐5 cells exposed to different concentrations of SAHA for 24, 48, and 72 h in the presence of TGF‐β1 (n = 3). (B, C) Fluorescence images and quantification of EdU incorporation assay in MRC‐5 cells co‐cultured with TGF‐β1 and different concentrations of SAHA for 24 h (n = 3). (D, E) Images and quantification of wound healing assay in MRC‐5 cells treated with TGF‐β1 and different concentrations of SAHA for 24 h (n = 3). The bars represent the mean ± SD of three separate experiments. * indicates p < 0.050, ** indicates p < 0.010, *** indicates p < 0.001, **** indicates p < 0.0001. # indicates p < 0.050, ## indicates p < 0.010, ### indicates p < 0.001.

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  FIGURE 3. SAHA increases mitochondrial ROS in MRC‐5 cells. (A) Fluorescence and bright‐field images of MRC‐5 cells treated with mito‐SOX red after exposure to TGF‐β1 and different concentrations of SAHA (n = 3). (B) Quantification of mean gray value of mito‐SOX (n = 3). The bars represent the mean ± SD of three separate experiments. * indicates p < 0.050, ** indicates p < 0.010, *** indicates p < 0.001.

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  FIGURE 4. SAHA increases the expression of p66Shc. (A, B, C, and D) Protein expression (both whole‐cell protein and mitochondrial protein) and mRNA expression of p66Shc in MRC‐5 cells treated with TGF‐β1 and different concentrations of SAHA (n = 3). (E, F) Immunofluorescence images and quantification of p66Shc (green) in MRC‐5 cells treated with TGF‐β1 and different concentrations of SAHA (n = 3). The bars represent the mean ± SD of three separate experiments. * indicates p < 0.050, ** indicates p < 0.010, *** indicates p < 0.001, **** indicates p < 0.0001.

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  FIGURE 5. Silencing p66Shc inhibits the differentiation of lung fibroblasts. (A, B, and C) Protein and mRNA expression of p66Shc in MRC‐5 cells transfected with either NC siRNA or p66Shc siRNA for 24 h (n = 3). (D, E, and F) Protein and mRNA expression of collagen I and α‐SMA in MRC‐5 cells transfected with either NC siRNA or p66Shc siRNA for 24 h in the presence of TGF‐β1 and SAHA (n = 3). (G, H, and I) Immunofluorescence images and quantification of collagen I (green) and α‐SMA (red) in MRC‐5 cells (n = 3). The bars represent the mean ± SD of three separate experiments. * indicates p < 0.050, ** indicates p < 0.010, *** indicates p < 0.001, **** indicates p < 0.0001.

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  FIGURE 6. Silencing p66Shc inhibits the proliferation and migration of lung fibroblasts. (A) Cell viability of MRC‐5 cells exposed to SAHA and TGF‐β1, and transfected with either NC siRNA or p66Shc siRNA for 24 h (n = 3). (B and C) Immunofluorescence images and statistical analysis of EdU incorporation assay in MRC‐5 cells transfected with either NC siRNA or p66Shc siRNA and co‐cultured with TGF‐β1 and SAHA (n = 3). (D and E) Images and statistical analysis of the wound healing assay in MRC‐5 cells transfected with either NC siRNA or p66Shc siRNA and treated with TGF‐β1 and SAHA (n = 3). The bars represent the mean ± SD of three separate experiments. * indicates p < 0.050, ** indicates p < 0.010, *** indicates p < 0.001, **** indicates p < 0.0001.

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  FIGURE 7. Histone modifications mediate the upregulation of p66Shc expression by SAHA. (A, B, C, and D) Acetylation and methylation modifications of histone H3 in lung fibroblasts after treatment with TGF‐β1 and SAHA (n = 3). (E) Modification of histone H3 in the region of the p66Shc gene after TGF‐β1 and SAHA treatment (n = 3). The bars represent the mean ± SD of three separate experiments. * indicates p < 0.050, ** indicates p < 0.010, *** indicates p < 0.001, **** indicates p < 0.0001.