Table 1.
Primers used for real-time reverse transcription-polymerase chain reaction analysis.
Fig 1.
Dexamethasone inhibits in vitro myofibroblastic differentiation of FSCs.
(A) FSCs seeded at 2000 cells/well in 96-well plates were treated with dexamethasone at the indicated concentrations for 7 days. The IC50-value was measured. (B) mRNA expression of α-SMA, Col3A1 and Col1A3 was analyzed by quantitative RT-PCR after treatment with dexamethasone 0, 0.2 and 2 uM for 14 days. (C) Immunofluorescence staining for α-SMA (red), types III (red) and I collagen (green) after treated with either dexamethasone 2uM or 0 uM (control) for 14 days. Bars = 50 μm. (D) The percentages of stained areas. (E) The percentages of myofibroblasts. Data are shown as mean ± SD (n = 3). Statistical significance is presented as **, p<0.01 compared with other groups. All experiments were repeated with FSCs isolated from three different donors.
Fig 2.
Dexamethasone inhibited FSCs formation of fibromatosis nodule in murine model.
(A–C) FSCs treated with 200 nM dexamethasone (Dex) for 3 days were then delivered with Matrigel, followed by transplantation beneath the dorsal skin of nude mice. (A) Macroscopic views of the transplants after 14 days in vivo. Scale = 1 mm. H&E staining and immunofluorescence staining for α-SMA, types III and type I collagen were performed. Bars = 50 μm. (B) The percentages of stained areas. (C) The percentages of myofibroblasts. (D–F) FSCs were delivered in Matrigel and transplanted under beneath the dorsal skin of nude mice. After 7 days, dexamethasone (2 mg/kg/day) dissolved in saline was injected subcutaneously daily for 1 week, and the control group received daily subcutaneous injections of 40 ml of saline alone for 1 week (D) Macroscopic views of the implants at 14 days of implantation in vivo. Scale = 1 mm. H&E staining and immunofluorescence staining for α-SMA, type III and type I collagen. Bars = 50 μm. (E) The percentages of stained areas. (F) The percentages of myofibroblasts. Data are shown as mean ± SD (n = 3). **, p<0.01 denotes statistical significance. All experiments were repeated with FSCs isolated from three different donors.
Fig 3.
Inhibition of TGF-β1 signaling, Smad family and down-regulation of TGF-β1 in in vitro dexamethasone-treated FSCs.
FSCs were treated with 0 μM, 0.2 μM, and 2 μM dexamethasone (Dex) for 3 days, followed by (A) and (B) Western blotting analysis of Smad family and Sp1. Immunoblotting of ß-actin & Smad2/3 was performed to show equal protein loading. (C) TGF-β1 protein levels, as measured by enzyme-linked immunosorbent assay (ELISA), in the conditioned media (D) quantitative RT-PCR analysis for mRNA expression of TGF-β1, where GAPDH was used as normalization control. (E) TGF-β1 protein levels between FSCs and BMSCs, as measured by enzyme-linked immunosorbent assay (ELISA), in the conditioned media. Data are shown as mean ± SD (n = 3). **, p<0.01 denotes statistical significance. All experiments were repeated with FSCs isolated from three different donors.
Fig 4.
Inhibition of fibromatosis nodule formation through TGF-β1 knockdown of FSCs.
Validation of knockdown efficiency by (A) quantitative RT-PCR analysis and (B) Western blotting for mRNA and protein expression of TGF-β1, respectively after transfection with a lentiviral vector carrying RNAi targeting TGF-β1 or non-targeting RNAi (CTR) for 2 days. (C–E) Transfection with a lentiviral vector carrying RNAi targeting TGF-β1 gene or non-targeting RNAi (CTR) for 14 days. (C) Immunofluorescence staining for α-SMA, type III and I collagen. FSCs were cultured for 14 days. Bars = 50 um. (D) The percentages of stained areas. (E) The percentages of myofibroblasts. (F–H) FSCs were transfected with a lentiviral vector carrying RNAi targeting TGF-β1 and non-targeting RNAi (CTR) were delivered with Matrigel and implanted beneath the dorsal skin of nude mice. Macroscopic views of the implants at 14 days of implantation in vivo. Scale = 1 mm. H&E staining and immunofluorescence staining for α-SMA, type III and type I collagen. Bars = 50 um. (G) The percentages of stained areas. (H) The percentage of myofibroblasts. Data are shown as mean ± SD (n = 3). **, p<0.01 denotes statistical significance. All experiments were repeated with FSCs isolated from three different donors.
Fig 5.
Treatment of TGF-β1 abolished TGF-β1 knockdown-mediated inhibition of myofibroblastic differentiation.
(A) Quantitative RT-PCR analysis for mRNA expression of α-SMA, type III and type I collagen genes. FSCs transfected with a lentiviral vector carrying RNAi targeting TGF-β1 or non-targeting RNAi (CTR) were cultured with either 10 ng/ml TGF-β1 or saline for 14 days. (B) Immunofluorescence staining for α-SMA, type III and I collagen. FSCs transfected with a lentiviral vector carrying RNAi targeting TGF-β1 or non-targeting RNAi (CTR) were cultured with either 10 ng/ml TGF-β1 or saline for 14 days. Bars = 50 um. (C) The percentages of stained areas. (D) The percentages of myofibroblasts. Data are shown as mean ± SD (n = 3). **, p<0.01 denotes statistical significance. All experiments were repeated with FSCs isolated from three different donors.