Journal: PLoS ONE

Article Title: Overexpression of Endoglin Modulates TGF-β1-Signalling Pathways in a Novel Immortalized Mouse Hepatic Stellate Cell Line

doi: 10.1371/journal.pone.0056116

Figure Lengend Snippet: ( A ) Cells were stimulated with indicated concentrations of TGF-β1 (0.1 ng/ml; 1.0 ng/ml), BMP-2 (25 ng/ml), PDGF-BB (25 ng/ml) or left untreated (Co.) for indicated time intervals from 10 min to 4 hrs. When indicated, the cells were treated with the ALK5 inhibitor SB431542 (5 µM). Cell extracts were prepared and cellular proteins tested for expression of PDGFRβ, phosphorylated Smad1/5/8 (pSmad1/5/8) and Smad2 (pSmad2) (I), the phosphorylated forms of p38 (p-p38) and ERK1/2 (pERK1/2), phosphorylated ATF-2 (pATF-2) (II), Id2 and GFP by Western blot. ( B ) Cells were stimulated for the indicated time intervals with EGF (50 ng/ml; 100 ng/ml). Thereafter, cell extracts were prepared and analyzed by Western blot using specific antibodies to linker phosphorylated Smad2 (pSmad2L), phosphorylated Smad1/5/8 (pSmad1/5/8) (I), the phosphorylated forms of p38 (p-p38) and ERK1/2 (pERK1/2), phosphorylated ATF-2 (pATF-2) (II) and GFP. Membranes ( A, B ) were incubated with an antibody specific for β-actin to demonstrate equal protein loading. Band intensities of the indicated proteins ( A, C ) were measured, normalized to β-actin and represented as fold induction of the unstimulated sample. Results show representative images of one of three independent experiments.

Article Snippet: When indicated, the cells were pre-treated for 30 min with 5 μM SB431542 (Tocris Bioscience, BIOZOL, Eching, Germany) or 1 μM Dorsomorphin (Biomol, Hamburg, Germany). respectively.

Techniques: Expressing, Western Blot, Incubation

Journal: PLoS ONE

Article Title: Overexpression of Endoglin Modulates TGF-β1-Signalling Pathways in a Novel Immortalized Mouse Hepatic Stellate Cell Line

doi: 10.1371/journal.pone.0056116

Figure Lengend Snippet: ( A ) Cells were stimulated for 30 min with TGF-β1 (0.1 ng/ml; 1.0 ng/ml), BMP-7 (25 ng/ml) or left untreated (Co.). When indicated, the cells were stimulated in the presence of SB431542 (SB, 5 µM) or Dorsomorphin (DM, 1 µM). Thereafter, protein extracts were analyzed by Western blot using specific antibodies to phosphorylated Smad1/5/8 (pSmad1/5/8), C-terminally-phosphorylated Smad2 (pSmad2), phosphorylated Smad3 (pSmad3), the phosphorylated forms of p38 (p-p38) and ERK1/2 (pERK1/2), CTGF, Id1, SV40-large T antigen (SV40), and GFP. ( B ) Cells were transiently transfected with the pSmad3-responsive luciferase reporter (CAGA) 12 -MLP-Luc. Thereafter, cells were stimulated with (0.1 ng/ml; 1.0 ng/ml) or without (0) TGF-β1 for 6 hrs in the presence or absence of SB431542 (5 µM). Cells were lysed and the luciferase activity determined, normalized to the protein content of the corresponding sample and expressed as fold induction to unstimulated control samples. ( C ) Cells were stimulated for 48 hrs with TGF-β1 (0.1 ng/ml; 1.0 ng/ml) or left untreated (Co.) and when indicated pre-incubated with SB431542 (SB, 5 µM) during starvation (∼16 hrs). Thereafter, a first sample was taken to monitor protein expression before stimulation (Co., 0 h). After the stimulation, protein extracts were analyzed for expression of Endoglin (with an antibody that is specific for mouse Endoglin), TGF-β receptor II (RII), Fibronectin, ColI, vimentin, α-SMA, CTGF, Id2, and GFP by Western blot. Membranes ( A, C ) were incubated with an antibody to β-actin to monitor equal protein loading . Band intensities of the indicated proteins ( A, C ) were measured, normalized to β-actin and represented as fold induction of the unstimulated sample. The depicted results show representative images of one of three independent experiments.

Article Snippet: When indicated, the cells were pre-treated for 30 min with 5 μM SB431542 (Tocris Bioscience, BIOZOL, Eching, Germany) or 1 μM Dorsomorphin (Biomol, Hamburg, Germany). respectively.

Techniques: Western Blot, Transfection, Luciferase, Activity Assay, Control, Incubation, Expressing

Journal: PLoS ONE

Article Title: Overexpression of Endoglin Modulates TGF-β1-Signalling Pathways in a Novel Immortalized Mouse Hepatic Stellate Cell Line

doi: 10.1371/journal.pone.0056116

Figure Lengend Snippet: ( A ) Cells were transiently transfected using Lipofectamine 2000 with a cDNA encoding rat endoglin. Thereafter, cells were stimulated with or without BMP-7 (25 ng/ml) for one hour or with PDGF-BB (25 ng/ml) for 10 min. Cellular proteins were prepared and analyzed in Western blot using specific antibodies to endoglin (PPabE2), PDGFRβ, phosphorylated Smad1/5/8 (pSmad1/5/8), phosphorylated ERK1/2 (pERK1/2) and GFP. ( B ) Cells were transiently transfected as described in ( A ) and stimulated or not with PDGF-BB (25 ng/ml) in the presence or absence of SB431542 (5 µM). Thereafter, cellular proteins were prepared and analyzed in Western blot using specific antibodies to endoglin (PPabE2), PDGFRβ, phosphorylated ATF-2 (pATF-2), phosphorylated ERK1/2 (pERK1/2) and GFP. All membranes were incubated with an antibody to β-actin to demonstrate equal protein loading. For densitometric analysis bands of β-actin and p-p42 were scanned and the latter one normalized to β-actin. Results are expressed as fold induction relative to the pcDNA control. Results show representative images of one of three independent experiments.

Article Snippet: When indicated, the cells were pre-treated for 30 min with 5 μM SB431542 (Tocris Bioscience, BIOZOL, Eching, Germany) or 1 μM Dorsomorphin (Biomol, Hamburg, Germany). respectively.

Techniques: Transfection, Western Blot, Incubation, Control