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

Journal: Archives of Toxicology

Article Title: BMP6 participates in the molecular mechanisms involved in APAP hepatotoxicity

doi: 10.1007/s00204-024-03954-5

Figure Lengend Snippet: Hepatic BMP6 expression is increased in an animal model of APAP-induced ALF. A Representative 4X and 10X images of H&E and percentage of necrotic area quantification (%). B ALT and AST activity determination in serum samples. C and D Hmox1 and Bmp6 mRNA levels determined by RT-qPCR and normalized to 36b4 gene expression. Data are expressed as fold of change relative to control condition (Control). E Correlation of Bmp6 mRNA expression with Hmox1 mRNA expression. F Representative 10X and 20X images of BMP6 immunostaining from liver sections and its quantification. Data are expressed as arbitrary units (a.u.). G Correlation of BMP6 hepatic expression with percentage of necrotic area. Experimental conditions: Mice i.p. injected with vehicle (saline) or APAP (300 mg/kg) and sacrificed 6 or 24 h after APAP administration ( n = 12 animals per group). ** p < 0.01, *** p < 0.005 and **** p < 0.0001, APAP 6 h or 24 h vs. Control

Article Snippet: Serum BMP6 concentration was quantified by ELISA using the mouse BMP6 ELISA kit (CSB-E09279m) for mouse samples and the human BMP6 ELISA kit (CSB-E09277h) for human samples (Cusabio Technology LLC, Hubei, China), following the manufacturer ́s instructions.

Techniques: Expressing, Animal Model, Activity Assay, Quantitative RT-PCR, Gene Expression, Control, Immunostaining, Injection, Saline

Journal: Archives of Toxicology

Article Title: BMP6 participates in the molecular mechanisms involved in APAP hepatotoxicity

doi: 10.1007/s00204-024-03954-5

Figure Lengend Snippet: Circulating BMP6 is increased in mice after APAP overdose and in patients with APAP intoxication. Experimental conditions: Mice i.p. injected with vehicle (saline) or APAP (300 mg/kg) and sacrificed 6 or 24 h after APAP administration ( n = 12 animals per group). A Serum levels of BMP6 determined by ELISA. Data are expressed as pg/ml. B and C Correlation of serum BMP6 levels with hepatic expression of BMP6, circulating ALT and AST, respectively. Study population: 18 patients with APAP overdose, 9 with DILI (ALT > 100 U/L) and 9 without DILI. D Serum levels of BMP6 determined by ELISA. Data are expressed as pg/ml. E Correlation of serum BMP6 levels with circulating ALT levels. ** p < 0.01 and *** p < 0.005, APAP 6 h or 24 h vs. Control

Article Snippet: Serum BMP6 concentration was quantified by ELISA using the mouse BMP6 ELISA kit (CSB-E09279m) for mouse samples and the human BMP6 ELISA kit (CSB-E09277h) for human samples (Cusabio Technology LLC, Hubei, China), following the manufacturer ́s instructions.

Techniques: Injection, Saline, Enzyme-linked Immunosorbent Assay, Expressing, Control

Journal: Archives of Toxicology

Article Title: BMP6 participates in the molecular mechanisms involved in APAP hepatotoxicity

doi: 10.1007/s00204-024-03954-5

Figure Lengend Snippet: Immortalized mouse hepatocytes showed elevated expression and release of BMP6 after APAP treatment. A Cell viability determined by crystal violet staining. Data are expressed as percentage relative to control condition (100%). B Cytotoxicity determined by lactate dehydrogenase (LDH) release. Data are expressed as percentage relative to the positive control (100%). C Representative 40X images of DAPI staining. D ROS production detected with DHE probe represented as DHE fluorescence F/F 0 (a.u.). E Representative blots of the cell lysates with the indicated antibodies. F Bmp6 mRNA levels determined by RT-qPCR and normalized to 36b4 gene expression. Data are expressed as fold of change relative to control condition. G Representative blot of the cultured media (CM) with BMP6 antibody. Ponceau staining was used as loading control. Experimental conditions: immortalized mouse hepatocytes treated with 1 mM or 5 mM APAP for 2 (D), 6 (E) or 16 (A, B, C, F, G) hours (N > 3 independent experiments). ** p < 0.01, *** p < 0.005 and **** p < 0.0001, 1 or 5 mM APAP vs. non-treated cells

Article Snippet: Serum BMP6 concentration was quantified by ELISA using the mouse BMP6 ELISA kit (CSB-E09279m) for mouse samples and the human BMP6 ELISA kit (CSB-E09277h) for human samples (Cusabio Technology LLC, Hubei, China), following the manufacturer ́s instructions.

Techniques: Expressing, Staining, Control, Positive Control, Fluorescence, Quantitative RT-PCR, Gene Expression, Cell Culture

Journal: Archives of Toxicology

Article Title: BMP6 participates in the molecular mechanisms involved in APAP hepatotoxicity

doi: 10.1007/s00204-024-03954-5

Figure Lengend Snippet: Huh7 hepatocytes release BMP6 after APAP exposure. A Cell viability determined by crystal violet staining. Data are expressed as percentage relative to control condition (100%). B Cytotoxicity determined by lactate dehydrogenase (LDH) release. Data are expressed as percentage relative to the positive control (100%). C Representative 40X images of DAPI staining. D ROS production detected with DHE probe represented as DHE fluorescence F/F 0 (a.u.). E Representative blots of the cell lysates with the indicated antibodies. F BMP6 mRNA levels determined by RT-qPCR and normalized to 36B4 gene expression. Data are expressed as fold of change relative to control condition. G Representative blot of the cultured media (CM) with BMP6 antibody. Ponceau staining was used as loading control. Experimental conditions: Huh7 treated with 10 mM and 20 mM APAP for 2 (D), 6 (E) or 16 (A, B, C, F, G) hours ( N > 3 independent experiments). **** p < 0.0001, 10 or 20 mM APAP vs. non-treated cells

Article Snippet: Serum BMP6 concentration was quantified by ELISA using the mouse BMP6 ELISA kit (CSB-E09279m) for mouse samples and the human BMP6 ELISA kit (CSB-E09277h) for human samples (Cusabio Technology LLC, Hubei, China), following the manufacturer ́s instructions.

Techniques: Staining, Control, Positive Control, Fluorescence, Quantitative RT-PCR, Gene Expression, Cell Culture

Journal: Archives of Toxicology

Article Title: BMP6 participates in the molecular mechanisms involved in APAP hepatotoxicity

doi: 10.1007/s00204-024-03954-5

Figure Lengend Snippet: Silencing of BMP6 in Huh7 cells does not affect APAP-induced cell death. Experimental conditions (A–E): siBMP6 and control (siC) Huh7 cells treated with 20 mM APAP for 16 h ( N > 3 independent experiments). A BMP6 mRNA levels determined by RT-qPCR and normalized to 36B4 gene expression. Data are expressed as fold of change relative to control condition (siC-C). B Representative blots of the cell lysates with the indicated antibodies and the corresponding quantification. Data are expressed as fold of change relative to control condition (siC). C Cell viability determined by crystal violet staining. Data are expressed as percentage relative to control condition (C, 100%). D Cytotoxicity determined by lactate dehydrogenase (LDH) release. Data are expressed as percentage relative to the positive control (100%). E Representative blots of the cell lysates with the indicated antibodies. F Experimental conditions (F–I): siBMP6 and control (siC) Huh7 cells treated with 20 mM APAP for 16 h. Huh7 cells incubated with siBMP6- or siC-CM for 24 h ( N > 3 independent experiments). G Representative blot of the cultured media (CM) with BMP6 antibody. Ponceau staining was used as loading control. H . Cell viability determined by crystal violet staining. Data are expressed as percentage relative to control condition (C, 100%). I Cytotoxicity determined by lactate dehydrogenase (LDH) release. Data are expressed as percentage relative to the positive control (100%). * p < 0.05, *** p < 0.005 and **** p < 0.0001, APAP vs. C; # p < 0.05 and ## p < 0.01, siBMP6 vs. siC

Article Snippet: Serum BMP6 concentration was quantified by ELISA using the mouse BMP6 ELISA kit (CSB-E09279m) for mouse samples and the human BMP6 ELISA kit (CSB-E09277h) for human samples (Cusabio Technology LLC, Hubei, China), following the manufacturer ́s instructions.

Techniques: Control, Quantitative RT-PCR, Gene Expression, Staining, Positive Control, Incubation, Cell Culture

Journal: Archives of Toxicology

Article Title: BMP6 participates in the molecular mechanisms involved in APAP hepatotoxicity

doi: 10.1007/s00204-024-03954-5

Figure Lengend Snippet: BMP6 promotes THP1 polarization toward an anti-inflammatory phenotype (M2). A Experimental conditions: siBMP6 and control (siC) Huh7 hepatocytes treated with 20 mM APAP for 16 h. THP1 cells incubated with siBMP6- or siC-CM for 24 h (N > 3 independent experiments). B, C, D and E mRNA levels of IL4, IL13, ARG1, MRC1, IL1B, IL6 and TNFA determined by RT-qPCR and normalized to 36B4 gene expression. Data are expressed as fold of change relative to the control condition (C or -). Experimental conditions: ( D and E ) THP1 treated with BMP6 (1, 10 and 100 ng/ml) for 24 h ( N > 3 independent experiments performed by duplicate). * p < 0.05, ** p < 0.01, *** p < 0.005 and **** p < 0.0001, APAP vs. C, or BMP6 vs. -; # p < 0.05 siBMP6 CM vs. siC CM

Article Snippet: Serum BMP6 concentration was quantified by ELISA using the mouse BMP6 ELISA kit (CSB-E09279m) for mouse samples and the human BMP6 ELISA kit (CSB-E09277h) for human samples (Cusabio Technology LLC, Hubei, China), following the manufacturer ́s instructions.

Techniques: Control, Incubation, Quantitative RT-PCR, Gene Expression