Journal: The Journal of Biological Chemistry

Article Title: Characterization of erythroferrone structural domains relevant to its iron-regulatory function

doi: 10.1016/j.jbc.2023.105374

Figure Lengend Snippet: ERFE and BMP receptors bind BMPs in a similar manner. A , crystallography-resolved structure of BMP2 homodimer bound to ALK3 (PDB: 2QJ9 ). Callout box enlarges the interaction and identifies the contacting residues. B , AlphaFold2 model of a BMP2 homodimer ( tan color) bound to ERFE colored by segment ( left ) and confidence pLDDT ( right ). Callout box shows the analogous interaction that BMPs have with their receptors. C , surface plasmon resonance sensorgrams of competition between WT ERFE, W82A ERFE, and activin RIIA for BMP2 ( left ) and BMP6 ( right ) binding. Extracellular portions of activin RIIA were immobilized, and BMP analyte flowed over alone or mixed with WT or W82A ERFE N-terminal segments. D , surface plasmon resonance of the ERFE N terminus binding to extracellular portions of five BMP receptors. BMP, bone morphogenetic protein; ERFE, erythroferrone; pLDDT, predicted local distance difference test.

Article Snippet: For the SPR competition assay, Activin RIIA-Fc (#340-RC2-100), BMPRIA/ALK3-Fc (#2406-BR-100), BMPRII-Fc (#811-BR-100), Activin RIA/ALK2-Fc (#637-AR-100), Activin RIIB-Fc (#339-RB-100/CF), human IgG1-Fc for control (#110-HG-100), BMP2/6 (#7145-BP-010/CF), BMP2 (#355-BM-010/CF), and BMP6 (507-BP-020/CF) were all obtained from RND Systems.

Techniques: SPR Assay, Binding Assay

Journal: Development (Cambridge, England)

Article Title: BMP signaling regulates satellite cell-dependent postnatal muscle growth

doi: 10.1242/dev.144089

Figure Lengend Snippet: Expression dynamics of BMP signaling pathway components in satellite cells during postnatal muscle growth. The relative mRNA copy numbers per 103 Gapdh mRNA copies of different BMP ligands (Bmp2, Bmp4, Bmp5, Bmp6, Bmp7, Bmp13 and Bmp14), BMP receptor type I Alk3, BMP target gene Id1, BMP antagonists Nog (encoding Noggin), Grem1 (encoding gremlin), Fst (encoding follistatin) and Chrd (encoding chordin) in satellite cells that were isolated by FACS from skeletal muscles of wild-type mice (n=3 biological and technical replicates) at P3, P14, P21 and P28. Data are mean±s.e.m.

Article Snippet: Where specified, BMP soluble receptor (recombinant mouse BMPR-IA/ALK3-Fc Chimera, R&D Systems, 437-MR) in a concentration of 200 ng/ml was added to the medium to block residual BMP signaling components.

Techniques: Expressing, Isolation, Muscles

Journal: Development (Cambridge, England)

Article Title: BMP signaling regulates satellite cell-dependent postnatal muscle growth

doi: 10.1242/dev.144089

Figure Lengend Snippet: Quantitative analysis following abrogation of BMP signaling in cultured Pax7CreERT2/+;RS6+/− satellite cell-derived primary myoblasts. Experimental protocol: at day 0, satellite cells from Pax7CreERT2/+ or Pax7CreERT2/+;RS6+/− adult mice were isolated using FACS and cultured in proliferation media; from days 2-4, cells remained either untreated (control), or were treated with 1 μM hydroxytamoxifen (4-OHT) or with 50 ng/ml of recombinant mouse Nog protein; at day 5, cells were either fixed for immunocytochemistry or collected for RNA extraction. (A,B) The dot plots (median indicated by the horizontal line) depict the relative mRNA copy numbers per 103 Gapdh mRNA of human SMAD6 (A) or of the BMP target gene Id1 (B) from cultured satellite cells isolated from Pax7CreERT2/+;RS6+/− mice. (C) Cells were cultured at low density for a proliferation assay for the comparison of non-treated versus treated satellite cells isolated by FACS from skeletal muscles of Pax7CreERT2/+ and Pax7CreERT2/+;RS6+/− mice. The number of cells per colony was counted from at least three wells per condition and per mouse (three mice); at least 50 colonies of cells per condition and per mouse were quantified. Data are shown as Whiskers-Tukey box plots. All P values were calculated using a t-test. (D-F) Following cultures of satellite cells from Pax7CreERT2/+;RS6+/− mice, the number of positive cells is given as a percentage of the total number of stained cells per colony following immunostaining against (D) myogenin (MyoG), (E) myosin heavy chain (MHC) and (F) Ki67. The quantification was performed on 13 to 20 colonies per culture (n=3 cultures, each derived from cells isolated from one mouse, total of n=3 mice). Data are shown as Whiskers-Tukey box plots. All P values were calculated using a t-test. In C-F, boxes indicate the interquartile range (IQR), the horizontal line indicates the median, whiskers indicate [1.5 × IQR] and dots indicate the outliers. (G,H) Dot plots (median indicated by the horizontal line) depict the relative mRNA copy numbers per 103 Gapdh mRNA of (G) p21 and (H) p57 from FACS-isolated and cultured satellite cells from Pax7CreERT2/+;RS6+/− mice. Cells remained either untreated (control, n=4) or were treated with 1 μM 4-OHT (n=3) or 50 ng/ml of recombinant mouse Nog protein (n=3).

Article Snippet: Where specified, BMP soluble receptor (recombinant mouse BMPR-IA/ALK3-Fc Chimera, R&D Systems, 437-MR) in a concentration of 200 ng/ml was added to the medium to block residual BMP signaling components.

Techniques: Cell Culture, Derivative Assay, Isolation, Control, Recombinant, Immunocytochemistry, RNA Extraction, Proliferation Assay, Comparison, Muscles, Staining, Immunostaining