Journal: bioRxiv

Article Title: The MuSK-BMP pathway maintains myofiber size in slow muscle through regulation of Akt-mTOR signaling

doi: 10.1101/2022.11.05.514105

Figure Lengend Snippet: (A, B), MuSK expression: (A) Unpermeabilized WT and ΔIg3-MuSK myoblasts cells were fixed, and cell surface MuSK was visualized by immunostaining with an antibody directed against the MuSK Ig2 domain (red). Note that comparable levels and distribution of immunostaining were observed in both genotypes. (B) WT and ΔIg3-MuSK myoblast cell lines express comparable levels of MuSK transcript as assessed by qRT-PCR. Data are means ± SEM from five biological replicates and two independent experiments. (C-G), BMP signaling: (C) WT and ΔIg3-MuSK myoblasts were treated with 20 ng/ml BMP4 for 15 min and immunostained for pSmad1/5. Note the increased intensity of pSmad staining in nuclei of BMP4-treated WT compared to ΔIg3-MuSK cells. Data are from three independent experiments with three biological replicates per group. (D) pSmad1/5 and total Smad1 in BMP4-treated cells (as in C) were assessed by Western blotting. (E) The level of BMP4-induced pSmad1/5, normalized to total protein, was reduced ΔIg3-MuSK as compared to WT cells. Data are means ± SEM of three biological replicates and independent Western blots (**** p < 0.0001, two-way ANOVA with Bonferroni’s multiple comparisons). (F) Cells were treated as in (C) and Car3 transcript levels were assessed. Data are mean ± SEM of 5-6 biological replicates per condition and replicated twice (**** p < 0.0001, *** p < 0.001, * p < 0.05, two-way ANOVA with Bonferroni’s multiple comparisons). (G) Cultured primary myotubes from WT and ΔIg3-MuSK mice were treated with 25 ng/ml BMP4 for 2 hr and levels of Wnt11 mRNA were measured by qPCR. Note that ΔIg3-MuSK myotubes show reduced Wnt11 expression in response to BMP4 stimulation. Data are means ± SEM from 6 biological replicates from two experiments. (**** p < 0.0001, two-way ANOVA with Bonferroni’s multiple comparisons). (H) Agrin signaling: WT and ΔIg3-MuSK cultured primary myotubes were treated with agrin for 16 hours. Acetylcholine receptors (AChR) were stained with α-bungarotoxin (red) and number of AChR clusters per myotube were counted (two-way ANOVA with Bonferroni’s multiple comparisons).

Article Snippet: All primary antibodies used were rabbit monoclonals obtained from Cell Signaling Tech: phospho-Smad1/5 (Ser463/465) (#9516), total Smad1 (D5957, #6944), phospho-4E-BP1 (Thr37/46) (236B4; #2855), and total 4E-BP1 (53H11, #9644).

Techniques: Expressing, Immunostaining, Quantitative RT-PCR, Staining, Western Blot, Cell Culture

Journal: bioRxiv

Article Title: The MuSK-BMP pathway maintains myofiber size in slow muscle through regulation of Akt-mTOR signaling

doi: 10.1101/2022.11.05.514105

Figure Lengend Snippet: (A) WT and ΔIg3-MuSK soleus muscle protein extracts were isolated for Western blotting of pSmad1/5, total Smad1, p-4EBP1, and total 4EBP1. Total protein stain was used as loading control and for protein normalization. Levels of pSmad1/5 and p4EBP1 were determined as a ratio to total Smad1 and total 4EBP1, respectively. Note that p-4EBP1 levels were reduced in ΔIg3-MuSK soleus compared to WT (C), but unchanged in TA (D). pSmad 1/5 levels in ΔIg3-MuSK soleus and TA were comparable to WT (E and F). Data are means ± SEM from five biological replicates. Data are means ± SEM from five biological replicates (*p < 0.05, unpaired two tailed Student’s t test).

Article Snippet: All primary antibodies used were rabbit monoclonals obtained from Cell Signaling Tech: phospho-Smad1/5 (Ser463/465) (#9516), total Smad1 (D5957, #6944), phospho-4E-BP1 (Thr37/46) (236B4; #2855), and total 4E-BP1 (53H11, #9644).

Techniques: Isolation, Western Blot, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: A Comprehensive Molecular Portrait of Human Urine-derived Renal Progenitor Cells

doi: 10.1101/602417

Figure Lengend Snippet: (A) Real-time PCR-based confirmation of down-regulation of CD133 and activated expression of BMP7 after CHIR stimulation. (B) JUN is a major hub of protein interaction networks of UdRPCs treated with CHIR. Based on the Biogrid database protein interaction networks were constructed from the set of the most highly regulated 40 genes either up- or down in the UdRPCs treated with CHIR. The selected genes used to connect to the network with interactions from the Biogrid database are marked in green, genes added as Biogrid interactions are marked in red. Induction of WNT leading to GSK3B inhibition is reflected by the connection of GSK3B to JUN and to AXIN2 which is connected to CTNNB1 (β-catenin) – these all downstream targets of GSK3B in the WNT-signaling pathway. (C) Community clustering of the network identified several communities: JUN (red), GSK3B/AXIN2/CTNNB1 (green), LATS2 (yellow), EGFR (pink). Black lines refer to edges within a community, red lines to edges between different communities. (D) Western blot analysis of the phosphorylated levels of SMAD 2/3 and SMAD 1/5/8 in undifferentiated and differentiated UF45, UM51 and UM27.

Article Snippet: The membranes were then blocked with 5% respective primary antibodies: Total Smad 1 (CST, 1:1000, TBS-T 5% BSA), phospho Smad 1/5/8 (CST, 1:1000, TBS-T 5% milk), Total Smad 2/3 (CST, 1:1000, TBS-T 5% BSA), and phospho Smad 2/3 (CST, 1:1000, TBS-T 5% milk).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Construct, Inhibition, Western Blot

Journal: Oncotarget

Article Title: Oncogenic features of the bone morphogenic protein 7 (BMP7) in pheochromocytoma

doi:

Figure Lengend Snippet: A. IHC or IF on adrenal medullary tissue from wild-type and mutant rats was performed using a rat anti-BMP7 antibody or an anti-P-Smad1/5/8 antibody, respectively. These tissues are representative of five tissues per animal group. Original magnification: 400× B. Plasma Bmp7 levels were measured in seven MENX mutant rats and seven wild-type rats fasted for 12 h. Measurements were performed using a rat Bmp7 ELISA-KIT. ***, P < 0.001.

Article Snippet: The primary antibodies used are: human anti-BMP7 (R&D Systems, USA; clone #164311; dilution 1:1000); rat anti-Bmp7 (#4693, 1:100), P-Smad1/5/8 (#9511, 1:50), Smad1 (#5753; 1:500), AKT (#9272; 1:500) and P-AKT (#9271; 1:500) all from Cell Signaling Technology (Danvers MA, USA); integrin β1 (Abcam, Cambridge, UK; #EP1041Y; dilution 1:500); anti-Myc tag (Clontech, St-Germain-en-Laye, France; #631206; dilution 1:500), α-Tubulin (Sigma-Aldrich, Hamburg, Germany; #T5168; dilution 1:1000).

Techniques: Mutagenesis, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Oncogenic features of the bone morphogenic protein 7 (BMP7) in pheochromocytoma

doi:

Figure Lengend Snippet: A. PC12 cells were transfected with a BMP7-containing (BMP7) or an empty (mock) vector. Twenty-four h post transfection IF was performed using specific antibodies targeting integrin β1 (1:400) or BMP7 (1:100). Cell nuclei were counterstained with DAPI. In parallel, the expression of integrin β1 and BMP7 proteins was determined by western blotting using specific antibodies. α-Tubulin was used as loading control. B. PC12 cells were either transfected (txBMP7) with a Myc-BMP7 plasmid or treated with 100 ng/mL recombinant human BMP7 (rhBMP7). After 24 h, cells were collected and analyzed by western blotting using antibodies against P-Smad1/5/8 and Smad1. α-Tubulin was used as a loading control. C. PC12 cells were treated with rhBMP7 for 5, 15, 30, 45, or 60 min. Western blot analysis was performed using specific antibodies raised against integrin β1, AKT and P-AKT. α-Tubulin was used as loading control. D. We co-transfected PC12 cells with BMP7 and scr-siRNA or siRNA- Itgb1 and 24 h later proliferation, migration and invasion were assessed. Values for cells with knockdown of Itgb1 were normalized against the values of scr-siRNA-transfected cells arbitrarily set to 100%. The experiments were performed two times with three technical replicates with similar results. For proliferation, the average of the 2 experiments is shown. For migration/invasion, five random fields of each test at × 400 magnification were counted (average ± SD). **, P < 0.01; ***, P < 0.001. In parallel, the levels of BMP7, integrin β1, P-AKT and AKT were assessed in the transfected cells by western blotting with specific antibodies as described above. α-Tubulin was used as a loading control. E. PC12 cells were treated with the indicated concentrations of the P-AKT inhibitor GSK690693 and western blot analysis was performed 24 h later to determine the expression of BMP7, integrin β1, P-AKT, and AKT. α-Tubulin was used as a loading control. F. Expression of BMP7, integrin β1, and P-AKT in 10 human primary PCCs and one human normal medulla (cont.) was assessed by western blotting using specific antibodies. α-Tubulin was used as a loading control.

Article Snippet: The primary antibodies used are: human anti-BMP7 (R&D Systems, USA; clone #164311; dilution 1:1000); rat anti-Bmp7 (#4693, 1:100), P-Smad1/5/8 (#9511, 1:50), Smad1 (#5753; 1:500), AKT (#9272; 1:500) and P-AKT (#9271; 1:500) all from Cell Signaling Technology (Danvers MA, USA); integrin β1 (Abcam, Cambridge, UK; #EP1041Y; dilution 1:500); anti-Myc tag (Clontech, St-Germain-en-Laye, France; #631206; dilution 1:500), α-Tubulin (Sigma-Aldrich, Hamburg, Germany; #T5168; dilution 1:1000).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Recombinant, Migration, Knockdown

Journal: Oncotarget

Article Title: Oncogenic features of the bone morphogenic protein 7 (BMP7) in pheochromocytoma

doi:

Figure Lengend Snippet: A. MTT cells were treated with DMH1 (3 μM or 5 μM) or with DMSO vehicle. Proliferation was assessed at the indicated times by the WST-1 assay. The experiments were performed two times with six technical replicates each, and are expressed as the mean ± SD. The values are normalized against those of DMSO-treated cells set to 100%. ***, P < 0.001. B. MTT cells treated as in A for 24 h were used for migration assays as indicated in Figure . The percentage of cells that migrated was normalized against the values of DMSO-treated cells arbitrarily set to 100%. The experiment was performed two times with three technical replicates with similar results. Five random fields of each test at ×400 magnification were counted (±SD). *, P < 0.05; **, P < 0.01. C. MTT cells were treated with DMH1 (3 μM or 5 μM) or with DMSO vehicle for 24 h. Proteins were then collected and probed for the expression of integrin β1, P-Smad1/5/8, Smad1, P-AKT and AKT. α-Tubulin was used as a loading control. D. Rat primary tumor cells were treated with DMH1 (3 μM and 5 μM) or DMSO for 72 h and cell viability was assessed using the WST-1 assay. Shown is the average of five independent cultures from five mutant rats (8 months of age). The values are normalized against those of DMSO-treated cells set to 100%. Data were analyzed independently with six technical replicates each and are expressed as the mean ± SD. ***, P < 0.001.

Article Snippet: The primary antibodies used are: human anti-BMP7 (R&D Systems, USA; clone #164311; dilution 1:1000); rat anti-Bmp7 (#4693, 1:100), P-Smad1/5/8 (#9511, 1:50), Smad1 (#5753; 1:500), AKT (#9272; 1:500) and P-AKT (#9271; 1:500) all from Cell Signaling Technology (Danvers MA, USA); integrin β1 (Abcam, Cambridge, UK; #EP1041Y; dilution 1:500); anti-Myc tag (Clontech, St-Germain-en-Laye, France; #631206; dilution 1:500), α-Tubulin (Sigma-Aldrich, Hamburg, Germany; #T5168; dilution 1:1000).

Techniques: WST-1 Assay, Migration, Expressing, Control, Mutagenesis

Journal: Cell Death & Disease

Article Title: IL-6 potentiates BMP-2-induced osteogenesis and adipogenesis via two different BMPR1A-mediated pathways

doi: 10.1038/s41419-017-0126-0

Figure Lengend Snippet: a hBMSCs were plated at a low density and treated with rhBMP-2, IL-6, IL-6/sIL-6R or a combination of IL-6/sIL-6R and rhBMP-2 for 30 min, and then immunofluorescence analysis of Smad1 was performed. Original magnification: 200×. b Postconfluent hBMSCs were treated with rhBMP-2 in the presence or absence of IL-6/sIL-6R and DMH1 for 60 min, and then immunoblotting analysis of pSmad1/5/8 was performed and normalized against β-actin. c Postconfluence hBMSCs were treated with rhBMP-2 in the presence or absence of IL-6/sIL-6R and DMH1 for 3 days, and then immunoblotting analysis of Runx2 was performed and normalized against β-actin. d – e hBMSCs were plated at a low density and treated with rhBMP-2 in the presence or absence of IL-6/sIL-6R and DMH1 for 3 days. Then, the postconfluent cells were cultured with ODM, and real-time PCR analysis of OPN ( d ) and OCN ( e ) was performed. * P < 0.05, ** P < 0.01, compared with untreated cells or the indicated groups

Article Snippet: Smad1 shRNA plasmids were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Immunofluorescence, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: IL-6 potentiates BMP-2-induced osteogenesis and adipogenesis via two different BMPR1A-mediated pathways

doi: 10.1038/s41419-017-0126-0

Figure Lengend Snippet: a Postconfluent hBMSCs were treated with rhBMP-2 in the presence or absence of IL-6/sIL-6R and DMH1 for 3 days, and then immunoblotting analysis of PPARγ and C/EBPα was performed and normalized against β-actin. b hBMSCs were cotransfected with plasmids containing Smad1 for 24 h, and then immunoblotting analysis of pSmad1/5/8 in the cell lysates was performed after a 60-min treatment (normalized against GAPDH, lower plane). c hBMSCs were transfected with plasmids expressing Smad1 shRNA or empty vectors. Then, the cells were treated with rhBMP-2 in the presence or absence of IL-6/sIL-6R for 3 days, and then immunoblotting analysis of PPARγ and C/EBPα was performed and normalized against β-actin. d – e hBMSCs were transfected with plasmids containing Smad1 shRNA or empty vectors. Then, the cells were treated with rhBMP-2 in the presence or absence of IL-6/sIL-6R for 3 days, and then the postconfluent cells were cultured with ADM, and real-time PCR analysis of PPARγ ( d ) and aP2 ( e ) was performed. * P < 0.05, ** P < 0.01, compared with untreated cells or the indicated groups

Article Snippet: Smad1 shRNA plasmids were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Western Blot, Transfection, Expressing, shRNA, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Developmental biology

Article Title: Zeb2 regulates the balance between retinal interneurons and Muller glia by inhibition of BMP–Smad signaling

doi: 10.1016/j.ydbio.2020.09.006

Figure Lengend Snippet: BMP-mediated activation of the Id1 promoter. (A) Schematic representation of the mouse Id1 promoter (−3500/+1000, ATG = 0) and comparison with the sequence cloned upstream of the luciferase (luc) gene used for the luciferase assay (Id1-luc). Green lines: Eboxes representing putative Zeb2-binding sites; red lines: putative BMP–Smad-binding elements (indicated as S1/5_1 to S1/5_3). The red box in Id1-luc is the BMP-responsive regulatory region (−1133/−996) in the Id1 promoter. (B) Zeb2 chromatin immunoprecipitation (ChIP) in P2 retinas (N = 2). The enrichment relative to input of the putative Zeb2- (E4, E1) or Smad-binding region (S1/5_1) quantified by PCR is indicated. amy, amylase (C) Scatter plot indicating mean and median fold-change (N = 5) of luciferase activity units of Id1 reporter described in BMP-treated N2a cells expressing either empty vector, wild-type Zeb2 or Zeb2(AxAx)2, compared to non-BMP-treated cells. Significance for differences between the groups was determined by one-way repeated measures ANOVA (F = 11.52, P = 0.0044). Tukey post-hoc test revealed significant differences in BMP-mediated activation between the empty and wild-type Zeb2 groups (P = 0.026), and the wild-type Zeb2 and Zeb2(AxAx)2 groups (P = 0.0041). *P < 0.05. (D) Protein location of the four point mutations in the Smadbinding domain of Zeb2(AxAx)2. (E) mRNA levels of Zeb2, Smad1, Smad5 and Id1 in differentiating murine embryonic stem cells (mESCs). (F) Smad1/5 ChIP and (G) Zeb2 ChIP in neural differentiated mESCs. Putative BMP–Smads::Smad4-binding regions are indicated as S1/5_1 to S1/5_3, while E-boxes are indicated as E1 and E4. BMP–Smads and Zeb2 are recruited in region E4 (−1287/−1147). (F, G) ChIP values are normalized against the input and using amylase (amy) as a negative control (set to 1).

Article Snippet: Sonicated material was brought to a final volume of 2 ml with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 17 mM Tris-HCl pH 8, 170 mM NaCl) and incubated with 15 μg anti-Zeb2 antibody (H260, Santa Cruz) or 10 μg anti-Smad1/5 antibody (D4G2, Cell Signaling Technologies) overnight at 4°C on a rotating wheel.

Techniques: Activation Assay, Sequencing, Clone Assay, Luciferase, Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Expressing, Plasmid Preparation, Negative Control