To decrease cardiac contractility after MTZ (ablated) or DMSO (control) treatments, fish were immediately incubated in egg water with 1 mg/ml of tricaine (Sigma, St. Louis, MO) or 10 μM of blebbistatin (Sigma, St. Louis, MO) for 12 hr and then washed three times for analyses. To inhibit BMP or Erbb2 signaling after MTZ (ablated) or DMSO (control) treatments, fish were immediately treated with 10 μM Dorsomorphin (DM) (Sigma, St. Louis, MO) or 5 μM AG1478 (Sigma, St. Louis, MO), respectively. Fish were treated for 24, 48, or 72 hr as indicated. To inhibit BMP, Erbb2 or Notch signaling after MTZ (ablated) or DMSO (control) treatments, fish were immediately treated with 10 μM Dorsomorphin (DM) (Sigma, St. Louis, MO), 5 μM AG1478 (Sigma, St. Louis, MO), or 10 mM DAPT (Sigma, St. Louis, MO) until indicated time for each experiment.
Dorsomorphin dm
Manufactured by Merck Group
Sourced in United States
Dorsomorphin (DM) is a small molecule compound that acts as an inhibitor of bone morphogenetic protein (BMP) signaling pathways. It functions by selectively blocking the activity of BMP type I receptors, such as ALK2, ALK3, and ALK6. Dorsomorphin can be utilized as a research tool to study the role of BMP signaling in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Sb431542, by Merck Group (3 mentions)
Essential 8, by Thermo Fisher Scientific (2 mentions)
Matrigel, by BD (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
N2 supplement, by Thermo Fisher Scientific (2 mentions)
Tricaine, by Merck Group (1 mentions)
Blebbistatin, by Merck Group (1 mentions)
Ag1478, by Merck Group (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Merck Group (1 mentions)
Basic fibroblast growth factor (bfgf), by Gemini Bio (1 mentions)
Essential 8 medium, by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Realtime glo mt cell viability assay kit, by Promega (1 mentions)
Filtermax f5 multi mode microplate reader, by Molecular Devices (1 mentions)
Bmp 2, by R&D Systems (1 mentions)
7 protocols using dorsomorphin dm
1
Cardiac and Signaling Pathway Modulation in Zebrafish
2
Differentiation of iPSCs into NPCs
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The use of human iPSCs was approved by the Institutional Review Board (IRB) of Yonsei University (Permit Number: 7001988-201802-BR-119-01E). We cultured iPSC on Matrigel (354277, BD Biosciences (San Jose, CA, United States)) with Essential 8 medium (A1517001, Invitrogen (Carlsbad, CA, United States)) coating [20 (link),21 (link),22 (link)]. For the differentiation of iPSC into NPC, an embryoid body (EB) was generated through the culturing of human iPSCs for 5–6 days on non-adherent petri dishes in Essential 8 (Invitrogen) with additional supplement 5 μM dorsomorphin (DM) (P5499, Sigma-Aldrich (St. Louis, MO, United States)) and 5 μM SB431542 (Sigma-Aldrich)). Then we attached EBs in the new culture dish coated with Matrigel (BD Biosciences) with neural induction medium comprised of DMEM/F12 medium (11320033, Invitrogen), 1× N2 supplement (17502048, Invitrogen), 1× nonessential amino acids (11140050, Invitrogen) for 6 days. When neural rosette formation appeared in the center of the EBs, NPCs were collected for analysis [23 (link),24 (link),25 (link)].
3
Isolation and Subculture of PSA-NCAM+ NPCs
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The isolation and subculture of PSA-NCAM-positive NPCs was performed in accordance with the protocol developed by Kim et al. [68 (link)]. Briefly, neural differentiation of embryoid bodies (EBs) derived from human embryonic stem cells (SNUhES31, Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul, Korea) was induced by 5 μM dorsomorphin (DM) (Sigma-Aldrich, St. Louis, MO, USA) and 5 μM SB431542 (SB) (Calbiochem, San Diego, CA, USA) in hESC medium deprived of bFGF (Gibco, Grand Island, NY, USA) for 4 days.
PSA-NCAM-positive cells were purified by MACS from expanded neural rosette cells as described previously [69 (link)]. Briefly, neural rosette cells treated with 10 μM Y27632 (Sigma-Aldrich) for 1 h were dissociated with Accutase (Invitrogen), and the cells (~1 × 108 cells) were incubated with anti-PSA-NCAM antibody conjugated with microbeads (Miltenyibiotec, Auburn, TX, USA) for 15 min at 4 °C. After washing, the positively labeled cells (NPCPSA-NCAM+) were isolated by positive MACS selection and replated on the culture dish at a density of 3.5 × 105 cells/cm2 in N2B27 medium or 1× N2, 0.5× B27, and 0.5× G21 supplement (Gemini Bio-Products, West Sacramento, CA, USA) (referred to as NBG medium) plus 20 ng/mL of bFGF.
PSA-NCAM-positive cells were purified by MACS from expanded neural rosette cells as described previously [69 (link)]. Briefly, neural rosette cells treated with 10 μM Y27632 (Sigma-Aldrich) for 1 h were dissociated with Accutase (Invitrogen), and the cells (~1 × 108 cells) were incubated with anti-PSA-NCAM antibody conjugated with microbeads (Miltenyibiotec, Auburn, TX, USA) for 15 min at 4 °C. After washing, the positively labeled cells (NPCPSA-NCAM+) were isolated by positive MACS selection and replated on the culture dish at a density of 3.5 × 105 cells/cm2 in N2B27 medium or 1× N2, 0.5× B27, and 0.5× G21 supplement (Gemini Bio-Products, West Sacramento, CA, USA) (referred to as NBG medium) plus 20 ng/mL of bFGF.
4
Differentiation of Human iPSCs to Neural Progenitor Cells
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The use of human iPSCs was approved by the Institutional Review Board (IRB) of Yonsei University (permit No. 7001988-201802-BR-119-01E). In this study, we used pre-constructed iPSC cell lines (Cat. No. C-004-5C; Gibco, USA). We cultured iPSC cell lines on Matrigel (BD Biosciences, USA) with Essential 8 medium (Invitrogen, USA) coating (Chen et al., 2011 (link); 2017 (link); Higuchi et al., 2015 (link)). To differentiate iPSC to NPC, an embryoid body (EB) was generated through culturing of human iPSCs for 5-6 days on non-adherent petri dishes in Essential 8 (Invitrogen) with additional supplement of 5 μM dorsomorphin (DM; Sigma-Aldrich, USA) and 5 μM SB431542 (Sigma-Aldrich). EBs were then attached to the new culture dish coated with Matrigel (BD Biosciences) with neural induction medium comprised of DMEM/F12 medium (Invitrogen), 1× N2 supplement (Invitrogen), 1× nonessential amino acids (Invitrogen) for 6 days. When neural rosette formation appears in the center of the EBs, NPCs were collected by using Pasteur pipet (Jin et al., 2019 (link); Seo et al., 2015 (link); Shin et al., 2017 (link)).
5
hPSC Differentiation using Dual Inhibitors
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DE differentiation was initiated when the hPSCs reached 70–80% confluency. H1 hESCs were treated with 3 μM CH (Cayman Chemical) and 1 μM Dorsomorphin (DM) (Sigma Aldrich) for 24 h. Cells were then cultured for another three days in a basal medium containing 0.05% HSA and 200 μg/mL ascorbic acid. H9 hESCs were treated with 3 μM CH and 1 μM DM for 24 h. Cells were then cultured for another two days in a basal medium with B-27 supplement.
6
Evaluating SY-LB-35, SY-LB-57, and BMP2 on C2C12 Cell Viability
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C2C12 cells (100 µL) were seeded in a 96-well plate at 5 × 105 cells/mL and incubated overnight in complete growth media to achieve 80% confluency followed by serum-starvation for 16–18 h. Next, the cells were treated with SS medium alone as a positive control, Triton X-100 (125 µM) as negative control, 1 nM to 1 mM SY-LB-35 or SY-LB-57 or 0.001 ng/mL to 100 ng/mL BMP2 (R&D Systems, Minneapolis, MN, USA) for 24 h. For BMP receptor inhibition, cell viability assays were carried out in presence or absence of Dorsomorphin (DM; 10 µM; Sigma Aldrich, St. Louis, MO, USA), a non-selective inhibitor of type I BMP receptor activity.
Next, reagents from the RealTime-Glo™ MT Cell Viability Assay Kit (Promega, Madison, WI, USA) were diluted in SS medium and used according to manufacturer’s protocol. Cellular luminescence was measured using the FilterMax F5 Multi-mode Microplate Reader (Molecular Devices, San Jose, CA, USA). Data from three independent experiments with individual experiments carried out in triplicate was determined and reported as a percentage of control.
Next, reagents from the RealTime-Glo™ MT Cell Viability Assay Kit (Promega, Madison, WI, USA) were diluted in SS medium and used according to manufacturer’s protocol. Cellular luminescence was measured using the FilterMax F5 Multi-mode Microplate Reader (Molecular Devices, San Jose, CA, USA). Data from three independent experiments with individual experiments carried out in triplicate was determined and reported as a percentage of control.
7
Neural Differentiation via Embryoid Body
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Neural differentiation was induced by transferring approximately 9 × 10 3 cells in 20 μL of hESC medium without bFGF and supplemented with 50 μM Y-27632, 5 μM dorsomorphin (DM) (Sigma-Aldrich), and 5 μM SB431542 (Sigma-Aldrich) onto the lid of a 100 mm petri dish followed by culture for 2 days as a hanging drop, to form embryoid bodies (EBs). The day of hanging-drop preparation was de ned as EB day 0. On EB day 2, the EBs were transferred to a 100-mm petri dish and cultured for an additional 4 days in suspension in medium with same composition. On EB day 6, the EBs were attached to a 60-mm tissueculture dish coated with Matrigel (BD Biosciencesplate, San Jose, CA, USA) and cultured in neural induction medium [1× N2 supplement (Life Technologies) and 1× nonessential amino acids (Life Technologies) in DMEM/F12 medium] for 4 days. The cells were analyzed on EB day 10.
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