HNECs were seeded on 24-well culture plates (200,000 cells/well) in 1 mL complete KSFM and incubated overnight. A pilot dose-response study with agonists included was first performed. Cells were subsequently cultured for 24 h with two different concentrations of TGF-β1, Activin A, BMP4 or Activin B (R&D Systems, Abingdon, United Kingdom) or cultured for 20 h with TGF-β1, Activin A, BMP4 or Activin B, after which TNF-α (R&D Systems) was added. Cell-free supernatants were analysed for cytokine release. Cell were analysed for Ki67 and ICAM1 expression using flow cytometry.
Activin b
Manufactured by R&D Systems
Sourced in United States, United Kingdom
Activin B is a recombinant protein produced by R&D Systems. It belongs to the transforming growth factor beta (TGF-β) superfamily of proteins. Activin B functions as a growth and differentiation factor.
Automatically generated - may contain errors
Lab products found in correlation
Activin a, by R&D Systems (9 mentions)
Activin ab, by R&D Systems (3 mentions)
Gdf11, by R&D Systems (3 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (3 mentions)
Follistatin, by R&D Systems (2 mentions)
Recombinant human tgf β1, by R&D Systems (2 mentions)
Sb431542, by Merck Group (2 mentions)
Tgf β1, by R&D Systems (2 mentions)
Gdf 8, by R&D Systems (2 mentions)
Lapatinib, by Selleck Chemicals (1 mentions)
Inhibin a, by R&D Systems (1 mentions)
Celltiter glo assay, by Promega (1 mentions)
Luciferase reporter gene assay kit, by Roche (1 mentions)
Genios pro instrument, by Tecan (1 mentions)
Hek293 cells, by Qiagen (1 mentions)
Dulbecco s modified eagle s medium ham s f12, by Thermo Fisher Scientific (1 mentions)
Prism software, by GraphPad (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Mg132, by Merck Group (1 mentions)
Tgf β, by R&D Systems (1 mentions)
14 protocols using activin b
1
Cytokine Release in HNECs
2
Dose-Dependent Drug Screening Assay
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Cells were seeded in 96-well plates. Twenty-four-h post-seeding the cells were treated in randomized triplicate with nine doses of each compound in 1:5 (single drug treatment) or 1:2 (combination drug treatment) serial dilution as previously described [20 (link), 21 (link)] or single dose indicated in figure legends. Drug responses for the initial experiments were measured as the GI50 value (dose required to inhibit growth by 50%) as previously described [20 (link), 21 (link)]. Later experiments made use of the GR50 metric instead. The GR50 measurement is closely related to GI50 and was developed to permit more reproducible drug response metrics [22 (link)]. We utilized an online GR50 calculator to determine drug response for these cells [22 (link)]. Drug responses are relative to the untreated condition for each cell line tested. For the serial dilution drug treatment, the highest drug doses were: Lapatinib, 10 µM; A-83-01, 10 µM; Follistatin, 500 ng/ml. Cell proliferation was estimated using the Cell Titer-Glo® assay (Promega) or imaged in an IncuCyte® Live-Cell Analysis instrument. Dose–response curves were plotted based on the cell confluence data. Lapatinib was purchased from Selleckchem; Follistatin, Activin A, Activin B, Activin AB, and Inhibin A were purchased from R&D Systems. All reagents were reconstituted according to manufacturer’s instructions.
3
TGF-β and Activin Signaling Modulation
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Recombinant human TGF-β1, activin A, activin B, activin AB, follistatin, and neutralizing monoclonal anti-TGF-β1,-β2,-β3 antibody (#MAB1835) were all from R&D Systems (Wiesbaden, Germany). The ALK4/5/7 inhibitor SB431542 was purchased from Calbiochem/Merck (Darmstadt, Germany) and dissolved in dimethylsulfoxide.
4
Assessing Erythroid Differentiation in Hematopoietic Cells
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Human BM CD34+ cells from healthy donors were plated in Methocult media to assess BFU-E and CFU-E growth (Supporting information) (Lopez-Holgado et al, 2005 (link)). Human CD36+ cells were cultured in liquid media to evaluate RAP-011 effects on erythroid differentiation (Figure S2). In some experiments, Activin A, Activin B and GDF11 (R&D Systems) were added to the cultures with each fresh addition of media, at concentrations of 30 ng/ml for Activins or 50 ng/ml for GDF11.
5
Studying Activin and GDF3 Signaling
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Following transfection, cells were rinsed once and then incubated in pretreatment media (DMEM high glucose, 0.5% fetal bovine serum, 1% NEAA, 1% GlutaMAX, 1% Na Pyruvate, 1% antibiotics) for 24 hours. Following pretreatment incubation, cells were rinsed in assay media (DMEM high glucose, 0.5% FAF-BSA, 1% NEAA, 1% GlutaMAX, 1% Na Pyruvate, 1% antibiotics) then treated with or without 10 μM of the ALK4/5/7 kinase inhibitor SB431542 (EMD Millipore catalog No. 616464) for 3 hours. Dimethyl sulfoxide was used as a vehicle control in untreated cells. At the end of the 3-hour incubation, the media were removed and fresh assay media were added containing 25 ng/mL Activin B (R&D Systems catalog No. 659-AB-005), 500-ng/mL GDF3 (R&D Systems catalog No. 5754-G3-010) or ligand-free media. A total of 10-μM SB431542 or dimethyl sulfoxide vehicle was coincubated with ligands where indicated. After 30 minutes of incubation, cells were rinsed briefly in ice-cold phosphate-buffered saline and plates were flash-frozen by floating on liquid nitrogen.
6
Activin A/B Inhibition Assay in HEK293 Cells
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Human embryonic kidney 293 (HEK293) cells stably expressing the SMAD‐binding element 12 (SBE12) luciferase system were purchased from Qiagen. They were seeded about 75,000 cells/well/100 μl Dulbecco's modified Eagle's medium/Ham's F12 (Life Technologies) with 10% fetal bovine serum (FBS) into a 96‐well plate. After overnight incubation at 37°C with 5% CO2, the medium was replaced with fresh medium containing 1% FBS. activin A or activin B antibody was serially diluted (1:3) in the medium containing 1% FBS to produce the following titration range: 0.01 ng/ml to 1771.5 ng/ml. A total of 50 μl of each concentration was then mixed with an equal volume of 30 ng/ml of activin A or activin B (R&D Systems) and incubated at room temperature for 30 min. After that, the mixture was added to individual wells and incubated for 24 h. Subsequently, cells were washed once with phosphate‐buffered saline and subjected to lysis. Luminescence was measured using a GeniosPRO instrument (Tecan) with substrate injection (Luciferase Reporter Gene Assay Kit; Roche). Relative luciferase units were measured, and IC50 curves were fitted using GraphPad Prism software (GraphPad Software).
Cell culture, microarray analysis, and quantitative real‐time polymerase chain reaction are described in theSupporting Information .
Cell culture, microarray analysis, and quantitative real‐time polymerase chain reaction are described in the
7
Cytokine Signaling in Cell Lines
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MyD88 knockout (KO) human embryonic kidney 293 cells (HEK293-I3A) were a kind gift from G. Stark (Department of Molecular Genetics, Lerner Research Institute, Cleveland, OH, United States) (Li et al., 1999 (link)). HEK293-I3A cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Wisent Inc., Montreal, QC, Canada). Huh7 human hepatoma cells (ATCC) were maintained in DMEM. HepG2 human hepatoma cells (ATCC) were maintained in Eagle’s minimal essential medium (EMEM; Wisent Inc.). Namalwa cells and Raji B cells were a kind gift from R. Bertrand and W. Mourad, respectively (Centre de Recherche du Centre Hospitalier de l’Université de Montréal, CRCHUM, Montréal, QC, Canada), and were cultured in Roswell Park Memorial Institute medium 1640 (RPMI1640; Wisent Inc.) Cell lines were supplemented with 10% fetal bovine serum (FBS; Wisent Inc.) and penicillin/streptomycin (Wisent Inc.), and were incubated at 37°C with 5% CO2. Cells were treated, where indicated, with 12.5, 25, and 50 ng/ml of either Activin B (R&D Systems, Minneapolis, MN, United States) or BMP6 (R&D Systems), or with 15 ng/ml of TGF-β (R&D Systems) for 24 hr. Huh7 cells were treated with 10 μM of the proteasome inhibitor MG132 (Sigma-Aldrich, St. Louis, MO, United States).
8
Hormone Assay Protocol Validation
9
Recombinant Myostatin and Binding Proteins
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Myostatin was expressed from a Chinese hamster ovary (CHO) cell line and purified using methods similar to those described in Thies et al. (2001) [23 (link)]. The construction, expression, and purification of ActRIIB-FC were previously described in Lee et al. [24 (link)]. Recombinant human GDF-11, Activin A, Activin B, Activin AB, and BMP-9 (bone morphogenetic protein-9) were purchased from R&D Systems (Minneapolis, MN, USA). Murine RK35 (mRK35) is a mouse monoclonal antibody against mature myostatin. It was generated from myostatin knockout mice immunized with human myostatin. This antibody was selected for humanization as described in Apgar et al. [21 (link)], resulting in the generation of domagrozumab.
10
Transwell and Scratch Assays for Cell Migration
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Cell migration was detected using the transwell assay and scratching wound healing assay as we previously reported 6 , 11 . For the transwell assay, in brief, 100 μl of 50,000 treated or untreated BMSCs were plated in the upper compartment. Then, 600 μl of serum‐free control medium with Activin B (10 ng/ml; R&D Systems, Minneapolis, MI) or not was added to the bottom chamber. After 24 hours, crystal violet was used to stain the cells that migrated to the lower chamber. Six visual fields (×20) were randomly selected to calculate the number of migrated cells in each group.
A scratch wound healing assay was adapted based on previously described methods11 , 14 . After the cells were transfected or treated, they were serum starved overnight. Then, the cells were detached and seeded in 12‐well plates at a density of 1 × 105 cells/well. After scratching, the cells were cultured in the maintain‐medium (DMEM with 1% FBS) with or without Activin B. Images were taken immediately and at 24 hours after scratching. Image‐Pro Plus 6.0 software was used to calculate the scratching area.
A scratch wound healing assay was adapted based on previously described methods
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