Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1

doi: 10.1152/ajplung.00079.2017

Figure Lengend Snippet: TGFβR1 mediates TGFβ-induced Smad1/5 phosphorylation in mPASMC. SB505124 (SB), an ALK4/5/7 inhibitor, prevented TGFβ-induced Smad1/5 phosphorylation, while dorsomorphin (DM), an ALK1/2/3/6 inhibitor, partly decreased TGFβ-induced Smad1/5 phosphorylation. Serum-starved cells were treated with 1 μM SB, 10 μM DM, or DMSO vehicle for 1 h before additional treatment with 2.5 ng/ml TGFβ1 or 20 ng/ml BMP4 for 1 h. Cell lysates were collected, and the protein expression level of the indicated Smads and GAPDH were detected using immunoblotting. Immunoblot images shown are representative of at least three independent experiments. For the densitometry analysis, n = 4 in each group; *P < 0.05.

Article Snippet: Recombinant human TGF-β1 and BMP4, mouse (m)ALK1-Fc (all obtained from R&D Systems), and the kinase inhibitors SB505124 (S4696; Sigma) dorsomorphin (ab144821; Abcam) and LDN212854 (SML0965; Sigma) were also used.

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1

doi: 10.1152/ajplung.00079.2017

Figure Lengend Snippet: ALK1 is expressed in mPASMC, mFibroblasts, and the mouse pup lung. A: ALK1 mRNA expression in the indicated adult and P10 mouse tissues and cells detected using RT-PCR. B: ALK1 protein expression in the indicated tissues and cell lysates detected using immunoblotting. Equal amounts of proteins were resolved using PAGE; effective protein transfer to the immunoblot membrane was demonstrated using Ponceau S staining. ALK1 protein expression data shown are representative of at least three independent experiments. C: ALK1 immunoreactivity (red) was detected in smooth muscle cells in pulmonary arteries (arrows), epithelial cells (arrowheads), interstitial cells (double arrows), and macrophages (*) in P10 mouse pup lungs, counterstained with hematoxylin. Representative images from three pups; scale bar is 25 µm long.

Article Snippet: Recombinant human TGF-β1 and BMP4, mouse (m)ALK1-Fc (all obtained from R&D Systems), and the kinase inhibitors SB505124 (S4696; Sigma) dorsomorphin (ab144821; Abcam) and LDN212854 (SML0965; Sigma) were also used.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Membrane, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1

doi: 10.1152/ajplung.00079.2017

Figure Lengend Snippet: ALK1 regulates TGFβ-stimulated pSmad1/5 nuclear localization in mPASMC. A: ALK1 immunoreactivity is detected on the surface of mPASMC unless the antibody is preadsorbed with solubilized extracellular ALK1 domain (mALK1-Fc). Live cells were reacted with an anti-mALK1 antibody, an IgG2b monoclonal rat antibody that was generated against an extracellular fragment of mouse ALK1, without and with previous exposure to mALK1-Fc, or rat IgG2b, washed, fixed, and then reacted with a fluorescently labeled secondary antibody and DAPI. Subsequently, epifluorescence microscopy was performed. Typical images of two or three independent studies are shown. B: pretreatment with an anti-mALK1 antibody inhibits TGFβ-mediated BMP R-Smad phosphorylation. Cells were treated with 0 or 15 µg/ml anti-mALK1 and then incubated with 0 or 2.5 ng/ml TGFβ1 for 1 h. Subsequently, the cells were fixed and pSmad1/5 immunoreactivity, and DAPI reactivity was assessed. In additional immunoreactivity control studies, the cells were reacted with an isotype control instead of the anti-pSmad1/5 antibody. The nuclear immunoreactivity intensity signal in a region of interest identified by the DAPI reactivity was quantified and normalized with the mean level detected in the control cells. n = 45 per group; *P < 0.05. Scale bars = 50 µm.

Article Snippet: Recombinant human TGF-β1 and BMP4, mouse (m)ALK1-Fc (all obtained from R&D Systems), and the kinase inhibitors SB505124 (S4696; Sigma) dorsomorphin (ab144821; Abcam) and LDN212854 (SML0965; Sigma) were also used.

Techniques: Generated, Labeling, Epifluorescence Microscopy, Incubation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1

doi: 10.1152/ajplung.00079.2017

Figure Lengend Snippet: ALK2 does not inhibit TGFβ-induced BMP R-Smad phosphorylation in mPASMC. Although LDN212854 (LDN), an ALK2 inhibitor, prevented BMP-mediated Smad1/5 phosphorylation, it did not prevent the phosphorylation of these Smads by TGFβ. Serum-starved cells were treated with 125 nM LDN for 1 h before additional treatment with 2.5 ng/ml TGFβ1, 20 ng/ml BMP4, or DMSO vehicle for 1 h. Cell lysates were collected, and then the expression level of indicated Smads and GAPDH were detected using immunoblotting. Immunoblot images shown are representative of at least three independent experiments. For the densitometry analysis, n = 4 in each group; *P < 0.05.

Article Snippet: Recombinant human TGF-β1 and BMP4, mouse (m)ALK1-Fc (all obtained from R&D Systems), and the kinase inhibitors SB505124 (S4696; Sigma) dorsomorphin (ab144821; Abcam) and LDN212854 (SML0965; Sigma) were also used.

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1

doi: 10.1152/ajplung.00079.2017

Figure Lengend Snippet: TGFβR1 mediates TGFβ-induced Smad1/5 phosphorylation in mPASMC. SB505124 (SB), an ALK4/5/7 inhibitor, prevented TGFβ-induced Smad1/5 phosphorylation, while dorsomorphin (DM), an ALK1/2/3/6 inhibitor, partly decreased TGFβ-induced Smad1/5 phosphorylation. Serum-starved cells were treated with 1 μM SB, 10 μM DM, or DMSO vehicle for 1 h before additional treatment with 2.5 ng/ml TGFβ1 or 20 ng/ml BMP4 for 1 h. Cell lysates were collected, and the protein expression level of the indicated Smads and GAPDH were detected using immunoblotting. Immunoblot images shown are representative of at least three independent experiments. For the densitometry analysis, n = 4 in each group; *P < 0.05.

Article Snippet: Recombinant human TGF-β1 and BMP4, mouse (m)ALK1-Fc (all obtained from R&D Systems), and the kinase inhibitors SB505124 (S4696; Sigma) dorsomorphin (ab144821; Abcam) and LDN212854 (SML0965; Sigma) were also used.

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1

doi: 10.1152/ajplung.00079.2017

Figure Lengend Snippet: TGFβ stimulates Smad1/5 phosphorylation in primary mouse pup (m)PASMC and in an adult rat PASMC line (CS54). Cells were serum-starved for 24 h and then treated with the indicated amounts of TGFβ1 or BMP4 for 1 h. Cell lysates were then collected, and the protein expression level of the indicated phospho- and total Smads and GAPDH were detected using immunoblotting.

Article Snippet: Recombinant human TGF-β1 and BMP4, mouse (m)ALK1-Fc (all obtained from R&D Systems), and the kinase inhibitors SB505124 (S4696; Sigma) dorsomorphin (ab144821; Abcam) and LDN212854 (SML0965; Sigma) were also used.

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1

doi: 10.1152/ajplung.00079.2017

Figure Lengend Snippet: TGFβ stimulates Smad1/5 phosphorylation in mouse pup lung fibroblasts (mFibroblasts) and in a fetal lung fibroblast cell line (RFL-6 cells). The cells were serum-starved for 24 h, treated with the indicated amounts of TGFβ1 or BMP4 for 1 h, and the protein expression levels of TGFβ and BMP R-Smads and GAPDH in soluble cell lysates were determined using immunoblotting.

Article Snippet: Recombinant human TGF-β1 and BMP4, mouse (m)ALK1-Fc (all obtained from R&D Systems), and the kinase inhibitors SB505124 (S4696; Sigma) dorsomorphin (ab144821; Abcam) and LDN212854 (SML0965; Sigma) were also used.

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1

doi: 10.1152/ajplung.00079.2017

Figure Lengend Snippet: TGFβ increases pSmad1/5 nuclear compartmentation in mPASMC. Serum-starved cells were treated with 2.5 ng/ml TGFβ1, 50 ng/ml BMP4, or DMSO control for 1 h. Then the cells were fixed and permeabilized, and pSmad1/5 was detected using antibodies and wide-field epifluorescence microscopy. 4,6-diamidino-2-phenylindole (DAPI) was used to identify the nuclear compartment. Uncalibrated fluorescence signals corresponding to pSmad1/5 immunoreactivity in nuclear regions of interest were obtained and then referenced to the average nuclear pSmad1/5 fluorescence signal for the control cells. Index bar is 50 µm long. n = ~100 cells in each group; *P < 0.05.

Article Snippet: Recombinant human TGF-β1 and BMP4, mouse (m)ALK1-Fc (all obtained from R&D Systems), and the kinase inhibitors SB505124 (S4696; Sigma) dorsomorphin (ab144821; Abcam) and LDN212854 (SML0965; Sigma) were also used.

Techniques: Epifluorescence Microscopy, Fluorescence

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1

doi: 10.1152/ajplung.00079.2017

Figure Lengend Snippet: TGFβR1 mediates TGFβ-induced Smad1/5 phosphorylation in mPASMC. SB505124 (SB), an ALK4/5/7 inhibitor, prevented TGFβ-induced Smad1/5 phosphorylation, while dorsomorphin (DM), an ALK1/2/3/6 inhibitor, partly decreased TGFβ-induced Smad1/5 phosphorylation. Serum-starved cells were treated with 1 μM SB, 10 μM DM, or DMSO vehicle for 1 h before additional treatment with 2.5 ng/ml TGFβ1 or 20 ng/ml BMP4 for 1 h. Cell lysates were collected, and the protein expression level of the indicated Smads and GAPDH were detected using immunoblotting. Immunoblot images shown are representative of at least three independent experiments. For the densitometry analysis, n = 4 in each group; *P < 0.05.

Article Snippet: Recombinant human TGF-β1 and BMP4, mouse (m)ALK1-Fc (all obtained from R&D Systems), and the kinase inhibitors SB505124 (S4696; Sigma) dorsomorphin (ab144821; Abcam) and LDN212854 (SML0965; Sigma) were also used.

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1

doi: 10.1152/ajplung.00079.2017

Figure Lengend Snippet: ALK2 does not inhibit TGFβ-induced BMP R-Smad phosphorylation in mPASMC. Although LDN212854 (LDN), an ALK2 inhibitor, prevented BMP-mediated Smad1/5 phosphorylation, it did not prevent the phosphorylation of these Smads by TGFβ. Serum-starved cells were treated with 125 nM LDN for 1 h before additional treatment with 2.5 ng/ml TGFβ1, 20 ng/ml BMP4, or DMSO vehicle for 1 h. Cell lysates were collected, and then the expression level of indicated Smads and GAPDH were detected using immunoblotting. Immunoblot images shown are representative of at least three independent experiments. For the densitometry analysis, n = 4 in each group; *P < 0.05.

Article Snippet: Recombinant human TGF-β1 and BMP4, mouse (m)ALK1-Fc (all obtained from R&D Systems), and the kinase inhibitors SB505124 (S4696; Sigma) dorsomorphin (ab144821; Abcam) and LDN212854 (SML0965; Sigma) were also used.

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1

doi: 10.1152/ajplung.00079.2017

Figure Lengend Snippet: ALK1 regulates TGFβ-stimulated pSmad1/5 nuclear localization in mPASMC. A: ALK1 immunoreactivity is detected on the surface of mPASMC unless the antibody is preadsorbed with solubilized extracellular ALK1 domain (mALK1-Fc). Live cells were reacted with an anti-mALK1 antibody, an IgG2b monoclonal rat antibody that was generated against an extracellular fragment of mouse ALK1, without and with previous exposure to mALK1-Fc, or rat IgG2b, washed, fixed, and then reacted with a fluorescently labeled secondary antibody and DAPI. Subsequently, epifluorescence microscopy was performed. Typical images of two or three independent studies are shown. B: pretreatment with an anti-mALK1 antibody inhibits TGFβ-mediated BMP R-Smad phosphorylation. Cells were treated with 0 or 15 µg/ml anti-mALK1 and then incubated with 0 or 2.5 ng/ml TGFβ1 for 1 h. Subsequently, the cells were fixed and pSmad1/5 immunoreactivity, and DAPI reactivity was assessed. In additional immunoreactivity control studies, the cells were reacted with an isotype control instead of the anti-pSmad1/5 antibody. The nuclear immunoreactivity intensity signal in a region of interest identified by the DAPI reactivity was quantified and normalized with the mean level detected in the control cells. n = 45 per group; *P < 0.05. Scale bars = 50 µm.

Article Snippet: Recombinant human TGF-β1 and BMP4, mouse (m)ALK1-Fc (all obtained from R&D Systems), and the kinase inhibitors SB505124 (S4696; Sigma) dorsomorphin (ab144821; Abcam) and LDN212854 (SML0965; Sigma) were also used.

Techniques: Generated, Labeling, Epifluorescence Microscopy, Incubation