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: Experimental & Molecular Medicine

Article Title: Hepatocyte toll-like receptor 4 mediates lipopolysaccharide-induced hepcidin expression

doi: 10.1038/emm.2017.207

Figure Lengend Snippet: LPS induces hepcidin expression by hepatocytes. ( a ) AML12 cells were treated with different concentrations of LPS for 24 h, and the expression of mouse hepcidin (Hamp1) mRNA was measured by quantitative real time polymerase chain reaction (qRT-PCR). ( b ) AML12 cells were treated with LPS (1 μg ml −1 ) for the designated times. ( c ) Expression of mRNA encoding inducible nitric oxide synthase (iNOS) and Hamp1 in AML12 cells treated with 1 μg ml −1 LPS for 24 h. ( d ) AML12 cells were treated for 12 h with BMP6 (20 ng ml −1 ), IL-6 (20 ng ml −1 ) or LPS (1 μg ml −1 ), and the expression of Hamp1 mRNA was measured by qRT-PCR. ( e ) Hepcidin concentration in cell culture medium from LPS (1 μg ml −1 )-treated AML12 cells. ( f ) Iron concentration in AML12 cells treated with LPS (1 μg ml −1 ) for 24 h.

Article Snippet: LPS ( Escherichia coli 026:B6, L2654, Sigma Aldrich, St Louis, MO, USA), BMP6 (6325-BM, R&D Systems, Minneapolis, MN, USA) and IL-6 (CYT-213, PROSPEC, Ness-Ziona, Israel) were dissolved in manufacturer-recommended solvents.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Concentration Assay, Cell Culture