Figure 5

TGF-β-induced ERK/mitogen-activated protein kinase signalling in Dupuytren's disease is inhibited by selective low-molecular-weight kinase inhibitors and BMP6. (A) Western blot analysis for phosphorylated Smad2 (P-Smad2), P-Smad1, P-ERK1/2, type I collagen, α-SMA, c-myc and total ERK1/2 using specific antibodies on pooled control (mixture 1 through 4) and Dupuytren's (mixture 1 through 4) fibroblasts treated with SB-431542 (20 μmol), mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD98059 (10 μmol) and rec. BMP6 (100 ng/mL) for 18 hours. (B) Western blot analysis for P-ERK1/2, α-SMA, c-myc and total ERK1/2 using specific antibodies on pooled Dupuytren's fibroblasts (mixture 1 through 4) treated with SB-431542 (20 μmol), MEK1 inhibitor PD98059 (10 μmol), PDGF receptor (STI571, also called imatinib mesylate), epidermal growth factor receptor (PKI166) and vascular endothelial growth factor receptor (PTK787/ZK 222584) kinase inhibitors (10 μmol) for 18 hours. (C) 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based proliferation assay with pooled Dupuytren's fibroblasts (mixture 1 through 4) treated for 4 days with SB-431542 (20 μmol) and/or the MEK1 inhibitor PD98059 (10 μmol). Absorbance at 490 nm was measured daily, and the proliferation rate is stated relative to untreated Dupuytren's fibroblasts (mixture 1 through 4) at day 0. (D) FPCL on pooled control (mixture 1 through 4) and Dupuytren's (mixture 1 through 4) fibroblasts treated with SB-431542 (20 μmol) and/or PD98059 (10 μmol) for 72 hours.