Figure 2.

Suppression of Safe prevents TGF-β-induced cardiac fibrosis in vitro. (A) mRNA expression of Safe, Col1a1, α-SMA and Bmp1 in TGF-β-untreated or TGF-β-treated cardiac fibroblasts after Safe knockdown (n=3). (B) Western blot analysis and relative densitometric quantification of COL1A1 and α-SMA protein levels in TGF-β-untreated or TGF-β-treated cardiac fibroblasts after Safe knockdown (n=3). (C) Representative images of immunofluorescence staining for α-SMA (n=5) and quantification of the relative cell areas of cardiac fibroblasts after treatments as indicated (n=40). The nuclei were counterstained with DAPI (blue). Scale bar indicates 50 μm. (D) Representative images of collagen gel contraction for 24 hours and quantification of collagen area inside the dashed circles (n=3). Scale bar indicates 0.5 cm. (E) BMP1 protein enzyme activity in the supernatant of cultured fibroblasts after indicated treatments (n=3). The excitation wavelength is 320 nm, and the emission wavelength is 405 nm. (F) ELISA assay of COL1A1 protein in the supernatant of cultured fibroblasts after indicated treatments (n=3). (G) CCK-8 assay of cardiac fibroblasts with TGF-β-untreated or TGF-β-treated showing repressed cell proliferation by Safe knockdown. The cell proliferation rate was expressed as optical density value at 450 nm (OD₄₅₀) wavelength (n=3). (H) Flow cytometry analysis showing decreased ratios of pH3-positive fibroblasts and myofibroblasts in the group of Safe knockdown (n=3). (I) Representative images of immunofluorescence staining for mitosis marker 5-ethynyl-2'-deoxyuridine (EdU, magenta) and DAPI (blue), PDGFR-α (red) was stained as a marker of fibroblasts. Scale bar indicates 50 μm. Right panel: Percent of EdU⁺ cells in PDGFR-α⁺ cells (n=25). Data are presented as mean ± SEM; Student's t-test or one-way ANOVA; *p < 0.05.