Research
Elevated transforming growth factor β and mitogen-activated protein kinase pathways mediate fibrotic traits of Dupuytren's disease fibroblasts
1 Department of Molecular Cell Biology and Centre for Biomedical Genetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands
2 Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, D-14195 Berlin, Germany
3 Department of Orthopedic Surgery, Academic Medical Center, Meibergdreef 9, 1100 DD, Amsterdam, The Netherlands
Fibrogenesis & Tissue Repair 2011, 4:14 doi:10.1186/1755-1536-4-14
Published: 28 June 2011Additional files
Additional file 1:
Quantification of total Smad and phosphorylated Smad (P-Smad) protein expression levels as depicted in Figure 1B. Quantification was performed by densitometric analysis using the Odyssey system (LI-COR Biosciences). All values are expressed relative to β-actin protein expression levels (C, Control; D, Dupuytren).
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Additional file 2:
Fibroblast-populated collagen lattice (FPCL) of controls' (1 through 4) and Dupuytren's patients' (1 through 4) fibroblasts treated with dimethyl sulfoxide (DMSO) (-) or 20 μmol SB-431542 (+) in the presence or absence of 100 ng/mL rec. bone morphogenetic protein 6 (BMP6). Corresponding images demonstrating the quantification of contraction in Figure 3D are shown.
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Additional file 3:
Quantitative PCR was used to determine the average expression of α-SMA, Smad1, Smad2, Smad3, PAI-1 (plasminogen activator inhibitor 1), fibronectin, CTGF, c-myc and COL1A2 mRNA from control (mixture 1 through 4) and Dupuytren's (mixture 1 through 4) fibroblasts relative to GAPDH mRNA expression in the presence or absence of SB-431542 (20 μmol) and/or the MEK1 inhibitor PD98059 (10 μmol) and/or 100 ng/mL BMP6 for 18 hours. All values are expressed relative to the average of the untreated control (1 through 4) mRNA values.
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Additional file 4:
Top: FPCL on control (mixture 1 through 4) and Dupuytren's fibroblasts (mixture 1 through 4) treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 nmol) in the presence or absence of rec. BMP6 (100 ng/mL) for 72 hours. Middle: Representative images of each condition are shown. Bottom: Western blot analysis of phosphorylated extracellular signal-regulated kinase 1/2 (P-ERK1/2) and α-smooth muscle actin (α-SMA) on primary control (mixture 1 through 4) and Dupuytren's fibroblasts (mixture 1 through 4) treated with TPA (100 nmol) in the absence or presence of rec. BMP6 (100 ng/mL) for 18 hours are depicted in the lower panel. β-actin was included as a loading control.
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Additional file 5:
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based proliferation assay of pooled control fibroblasts treated for four days with SB-431542 (20 μmol) and/or the mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD98059 (10 μmol) where indicated. Absorbance at 490 nm was measured daily, and the proliferation rate is stated relative to untreated cells at day 0.
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