Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein (BMP) and Activin Type II Receptors Balance BMP9 Signals Mediated by Activin Receptor-like Kinase-1 in Human Pulmonary Artery Endothelial Cells

doi: 10.1074/jbc.m109.002881

Figure Lengend Snippet: FIGURE 1. BMP9 selectively activates mRNA transcription in HPAECs. A, confluent serum-restricted HPAECs were treated with BMP2, BMP4, BMP6, BMP9 (10 ng/ml), or TGF1 (2 ng/ml) in M199, 0.1% FBS (0.1%) for 1, 4, or 8 h. Total RNA was extracted and cDNA prepared. The expression of Id1, Id2, IL-6, IL-8, E-selectin, L-selectin, P-selectin, and fibroblast growth factor 2 (FGF2) were determined by qPCR, with expression being normalized to -actin and expressed as the fold-change relative to 0.1% at that time point. Data are presented as the mean S.E. of three experiments. *, p 0.05; **, p 0.01; or ***, p 0.001 compared with 0.1% FBS (0.1%). B, serum-restricted HPAECs were treated with BMP9 (1 ng/ml) in M199, 0.1% FBS (0.1%) for 0.5, 1, 2, and 4 h. Immunoblotting was performed with antibodies against Id1 and Id2. All blots were reprobed for -actin to ensure equal loading. C, HPAECs were treated with BMP9 (1 ng/ml) in M199, 0.1% FBS (0.1%) for 24 h. Conditioned medium was assayed for IL-8 using a specific enzyme-linked immunosorbent assay and data are expressed as picograms of IL-8/105 cells. Data are mean S.E. (n 6) from a representative experiment from 3 repeats.

Article Snippet: Western Blotting—Cells were grown to confluence in 6-cm dishes and serum-restricted in M199, 0.1% FBS for 16 h. Cells were then treated with recombinant human BMP2, BMP4, BMP6, BMP9, or TGF 1 (R&D Systems) diluted in 0.1% for 1 h. For ALK5 inhibition experiments, cells were preincubated for 30minwith SD208 (TGF Receptor kinase inhibitor V, Calbiochem) or dimethyl sulfoxide vehicle control in 0.1%, prior to treatment with ligands.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Bone Morphogenetic Protein (BMP) and Activin Type II Receptors Balance BMP9 Signals Mediated by Activin Receptor-like Kinase-1 in Human Pulmonary Artery Endothelial Cells

doi: 10.1074/jbc.m109.002881

Figure Lengend Snippet: FIGURE 2. BMP9 induction of Smad phosphorylation and mRNA tran- scription in HPAECs is concentration-dependent. A, serum-restricted HPAECs were treated with BMP9 (0.01–10 ng/ml), BMP2 (10 or 50 ng/ml), BMP4 (10 or 50 ng/ml), BMP6 (10 or 50 ng/ml), or TGF1 (2 or 5 ng/ml) in M199, 0.1% FBS (0.1%) for 1 h. Immunoblotting was performed with anti- bodies against phospho-Smad1/5, Smad1, phospho-Smad2, Smad2, phospho-Smad1/3, or Smad3. All blots were reprobed for -actin to ensure equal loading. B, serum-restricted HPAECs were treated with BMP9 (0.01–10 ng/ml) in 0.1% for 8 h. Total RNA was extracted and cDNA pre- pared. The expression of Id1, Id2, IL-8, and E-selectin were determined by qPCR, with expression being normalized to -actin and expressed as the fold-change relative to 0.1%. Data are presented as the mean S.E. of three experiments. C, serum-restricted HPAECs, HAECs, HMEC-1, and HPASMCs were treated with BMP9 (1 ng/ml) or TGF1 (5 ng/ml) in M199,

Article Snippet: Western Blotting—Cells were grown to confluence in 6-cm dishes and serum-restricted in M199, 0.1% FBS for 16 h. Cells were then treated with recombinant human BMP2, BMP4, BMP6, BMP9, or TGF 1 (R&D Systems) diluted in 0.1% for 1 h. For ALK5 inhibition experiments, cells were preincubated for 30minwith SD208 (TGF Receptor kinase inhibitor V, Calbiochem) or dimethyl sulfoxide vehicle control in 0.1%, prior to treatment with ligands.

Techniques: Phospho-proteomics, Concentration Assay, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Iron overload inhibits BMP/SMAD and IL-6/STAT3 signaling to hepcidin in cultured hepatocytes

doi: 10.1371/journal.pone.0253475

Figure Lengend Snippet: Huh 7 cells were treated with 100 μM DFO for 18 hours before washing and supplementation with 50 μM FAC. After 2 hours, cells were treated with 20 ng/ml human IL-6, 5 ng/ml BMP6, or both over 4 hours. (A) qPCR analysis of HAMP mRNA. (B) Western blot analysis of pSTAT3, STAT3, pSMAD5, SMAD5, ferritin and β-actin. (C-F) qPCR analysis of ID1 , TFRC , GCLC and HMOX1 mRNAs. All data in graphs are presented as the mean ± SEM from three independent experiments for (A) and two independent experiments for (B-F). Statistical analysis was performed by one-way ANOVA. Statistically significant differences (p<0.05) across iron treatments (compared to respective values from iron-unperturbed cells shown in black bars) are indicated by *.

Article Snippet: Following recombinant proteins were used: murine IL-6 (#5216, Cell Signaling), human IL-6 (#I1395, Sigma-Aldrich), human BMP6 (#507-BP, R&D Systems), and human holo-transferrin (#T4132, Sigma).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Iron overload inhibits BMP/SMAD and IL-6/STAT3 signaling to hepcidin in cultured hepatocytes

doi: 10.1371/journal.pone.0253475

Figure Lengend Snippet: (A) Huh 7 cells were pretreated with either 50 μM FAC, 50 μM FeSO 4 , or 30 μM holo-transferrin (HTF) for 2 hours and then treated with BMP6 for 4 hours; HAMP mRNA was measured by qPCR. (B) Huh 7 cells were treated with 100 μM SIH for 18 hours before washing and supplementation with 50 Fe-SIH. After 2 hours, cells were treated with 20 ng/ml IL-6, 5ng/ml BMP6, or both over 4 hours. HAMP mRNA was measured by qPCR. All data in graphs are presented as the mean ± SEM from three independent experiments. Statistical analysis was performed by one-way ANOVA. Statistically significant differences (p<0.05) across iron treatments (compared to respective values from iron-unperturbed cells shown in black bars) are indicated by *.

Article Snippet: Following recombinant proteins were used: murine IL-6 (#5216, Cell Signaling), human IL-6 (#I1395, Sigma-Aldrich), human BMP6 (#507-BP, R&D Systems), and human holo-transferrin (#T4132, Sigma).

Techniques:

Journal: PLoS ONE

Article Title: Iron overload inhibits BMP/SMAD and IL-6/STAT3 signaling to hepcidin in cultured hepatocytes

doi: 10.1371/journal.pone.0253475

Figure Lengend Snippet: Huh7 cells were treated with increasing doses of FAC over 2 hours. When indicated, the experiment was either terminated or the cells were washed and further incubated with 25 ng/ml BMP6 (A and C) or 5 ng/ml BMP6 (B, D-F) for 4 hours. (A and B) qPCR analysis of HAMP and ID1 mRNAs. (C) Western blot analysis of pSMAD5, SMAD5 and β-actin. (D-F) qPCR analysis of HAMP , ID1 and GCLC mRNAs. All data in graphs are presented as the mean ± SEM from three independent experiments. Statistical analysis was performed by one-way ANOVA (A, B, D and E) two-way ANOVA (F). Statistically significant differences (p<0.05) across iron treatments (compared to respective values from iron-unperturbed cells shown in black bars) are directly shown or indicated by *.

Article Snippet: Following recombinant proteins were used: murine IL-6 (#5216, Cell Signaling), human IL-6 (#I1395, Sigma-Aldrich), human BMP6 (#507-BP, R&D Systems), and human holo-transferrin (#T4132, Sigma).

Techniques: Incubation, Western Blot

Journal: PLoS ONE

Article Title: Iron overload inhibits BMP/SMAD and IL-6/STAT3 signaling to hepcidin in cultured hepatocytes

doi: 10.1371/journal.pone.0253475

Figure Lengend Snippet: Primary murine hepatocytes were maintained in serum-free William’s Medium E over the course of experiments. The cells were pretreated with 100 μM DFO for 18 hours and then washed and supplemented with 50 μM FAC over 2 hours. The cells were then treated with 20 ng/ml murine IL-6, 25 ng/ml BMP6, or both over 4 hours. (A) qPCR analysis of Hamp mRNA. (B) Western blotting of pSTAT3, STAT3, pSMAD5, SMAD1, ferritin, and β-actin. (C-F) qPCR analysis of Id1 , Smad7 , Socs3 and Tfrc mRNAs. All data in graphs are presented as the mean ± SEM from two independent experiments. Statistical analysis was performed by one-way ANOVA. Statistically significant differences (p<0.05) across iron treatments (compared to respective values from iron-unperturbed cells shown in black bars) are indicated by *.

Article Snippet: Following recombinant proteins were used: murine IL-6 (#5216, Cell Signaling), human IL-6 (#I1395, Sigma-Aldrich), human BMP6 (#507-BP, R&D Systems), and human holo-transferrin (#T4132, Sigma).

Techniques: Western Blot