Journal: EMBO Molecular Medicine

Article Title: Supraphysiological levels of GDF 11 induce striated muscle atrophy

doi: 10.15252/emmm.201607231

Figure Lengend Snippet: Relative p‐SMAD2/3 (left) and p‐SMAD1/5/8 (right) response, evaluated by AlphaLISA signal, of HEK293T cells after 1‐h exposure to recombinant myostatin (Mstn), activin A, GDF11, TGFβ, and BMP2. Relative p‐SMAD2/3 response of C2C12 myoblasts (left) and myotubes (right) after 1‐h exposure to Mstn, activin A, GDF11, and TGFβ. Phosphorylation of SMAD2 and SMAD3 in differentiated C2C12 myotubes following stimulation with recombinant Mstn or GDF11 for 30 and 60 min, as detected by immunoblotting. Equal loading is verified by Ponceau Red staining. EC 50 values (in nM) for p‐SMAD2/3 and p‐SMAD1/5/8 responses of HEK293T, C2C12 myoblasts, and C2C12 myotubes to the above listed ligands, as well as p‐SMAD1/5/8 response to BMP4, BMP6, and BMP7. Data information: Values are displayed as mean ± SEM; n = 4 for all data points.

Article Snippet: To assess the phosphorylation of SMAD in response to TGFβ superfamily members in undifferentiated cells, C2C12 myoblasts (ATTC CRL‐1772; passage 14) and HEK293T cells (ATCC CRL‐11268; passage 13) were plated on 96‐well tissue culture‐treated plates (Greiner‐Bio‐One 655098) in growth medium [high glucose Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 1% penicillin streptomycin (PS)], grown to confluency, and were treated with various doses of the following recombinant ligands for 1 h ( n = 4): Mstn (R&D Systems #788‐G8), GDF11 (R&D Systems # 1958‐GD), TGFβ (R&D Systems #240‐B), activin A (R&D Systems # 338‐AC), BMP‐2 (R&D Systems # 355‐BM), BMP‐4 (R&D Systems # 314‐BP), BMP‐6 (R&D Systems # 507‐BP), or BMP‐7 (R&D Systems # 354‐BP).

Techniques: Recombinant, Phospho-proteomics, Western Blot, Staining

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: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: A MicroRNA-124 Polymorphism is Associated with Fracture Healing via Modulating BMP6 Expression.

doi: 10.1159/000475570

Figure Lengend Snippet: Fig. 2. BMP6 as the candidate target gene ofmiR- 124-3p in cells with the ‘seed sequence’ in the 3’UTR.

Article Snippet: TBST containing 5% non-fat dry milk (Merck, Darmstadt, Germany) was used to block the membranes for 60 min. primary antibody against BMP6 (1:1,000; Santa Cruz Biotechnology, Beijing, China) was used to hybridize the membranes at 4°C for 12 hours.

Techniques: Sequencing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: A MicroRNA-124 Polymorphism is Associated with Fracture Healing via Modulating BMP6 Expression.

doi: 10.1159/000475570

Figure Lengend Snippet: Fig. 4. The correlation between the expression level of miR-124-3p and BMP6 mRNA.

Article Snippet: TBST containing 5% non-fat dry milk (Merck, Darmstadt, Germany) was used to block the membranes for 60 min. primary antibody against BMP6 (1:1,000; Santa Cruz Biotechnology, Beijing, China) was used to hybridize the membranes at 4°C for 12 hours.

Techniques: Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: A MicroRNA-124 Polymorphism is Associated with Fracture Healing via Modulating BMP6 Expression.

doi: 10.1159/000475570

Figure Lengend Snippet: Fig. 5. The expression of BMP6 mRNA (Fig. 5A) and protein (Fig. 5B) decreased in GG group compared with GC and CC group (** P<0.01 compared with the control).

Article Snippet: TBST containing 5% non-fat dry milk (Merck, Darmstadt, Germany) was used to block the membranes for 60 min. primary antibody against BMP6 (1:1,000; Santa Cruz Biotechnology, Beijing, China) was used to hybridize the membranes at 4°C for 12 hours.

Techniques: Expressing, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: A MicroRNA-124 Polymorphism is Associated with Fracture Healing via Modulating BMP6 Expression.

doi: 10.1159/000475570

Figure Lengend Snippet: Fig. 3. Luciferase activity reporter assay was conducted to verify BMP6 as the direct target gene of miR-124-3p (** P<0.01 compared with the control).

Article Snippet: TBST containing 5% non-fat dry milk (Merck, Darmstadt, Germany) was used to block the membranes for 60 min. primary antibody against BMP6 (1:1,000; Santa Cruz Biotechnology, Beijing, China) was used to hybridize the membranes at 4°C for 12 hours.

Techniques: Luciferase, Activity Assay, Reporter Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Berberine and aspirin prevent traumatic heterotopic ossification by inhibition of BMP signalling pathway and osteogenic differentiation

doi: 10.1111/jcmm.17919

Figure Lengend Snippet: The effect of berberine and aspirin on the BMP signalling pathway. C3H10T1/2 cells were treated for 24 h with only berberine or aspirin with different doses (A, B), or followed by BMP6 (20 ng/mL) for 30 min (C, D). Western blot was performed with indicated antibodies. Each experiment was repeated three times independently and the representative blots were shown in the figure.

Article Snippet: Recombinant mouse BMP‐6 protein was obtained from R&D System.

Techniques: Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Berberine and aspirin prevent traumatic heterotopic ossification by inhibition of BMP signalling pathway and osteogenic differentiation

doi: 10.1111/jcmm.17919

Figure Lengend Snippet: The combination effects of berberine and aspirin on the BMP signalling pathway. (A, B). C3H10T1/2 cells were treated with berberine and aspirin alone or in combination for 24 h. (C, D). The cells were treated with berberine and aspirin alone or in combination for 24 h, followed by BMP6 (20 ng/mL) for 30 min. Western blot was performed with indicated antibodies. Each experiment was repeated three times independently and the representative blots were shown in the figure.

Article Snippet: Recombinant mouse BMP‐6 protein was obtained from R&D System.

Techniques: Western Blot

Journal: The EMBO journal

Article Title: Self-organized developmental patterning and differentiation in cerebral organoids

doi: 10.15252/embj.201694700

Figure Lengend Snippet: Antibodies used in the study

Article Snippet: BMP6 , Rabbit , Novus Biologicals , NBP1‐19733 , 1:100.

Techniques: