mEpiSCs dissociated with Accumax were plated on MEF feeders in Ndiff 227 medium supplement with FGF2 (12 ng/mL) and Activin A (20 ng/mL) for 1 day (d-1-d0). The medium was changed to Basal medium and the various combinations of reagents shown in Figure 1 A and Table 1 at d0 for 16 days (d0-d16). The AMPK activator used was AICAR (1 mM, dilution in D2W, WAKO), A769662 (50 μM, dilution in DMSO, ADooQ), or metformin (1 mM, dilution in D2W, TCI). The medium was changed every 2 days. Because AICAR has some growth inhibitory effects (Chae et al., 2012 (link)) and the cell number decreased after long-time culturing with AICAR, in the first 6 days (d0-d6), the concentration of AICAR was 0.5 mM. After 16 days, the cells were dissociated with Accumax for further analysis or expansion on gelatin-coated 6-well plates in Basal medium or Ndiff227 medium with 2iL. The medium was replaced every other day and passaged every 4–7 days for at least 3 passages until stable colonies developed. A p38 inhibitor, SB203580 (10 μM, WAKO) was added 1–2 hr before AMPK activator treatment.
Sb203580
Manufactured by Fujifilm
Sourced in Japan
SB203580 is a pyridinyl imidazole compound that acts as a specific inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) enzyme. This laboratory product is used for research purposes to study the role of p38 MAPK signaling in various biological processes and disease models.
Automatically generated - may contain errors
Lab products found in correlation
Sp600125, by Fujifilm (6 mentions)
U0126, by Fujifilm (5 mentions)
Pd98059, by Fujifilm (3 mentions)
Bay11 7082, by Fujifilm (2 mentions)
Recombinant murine noggin, by Thermo Fisher Scientific (1 mentions)
Gsmtx4, by Abcam (1 mentions)
Yoda1, by Abcam (1 mentions)
U0126, by Cell Signaling Technology (1 mentions)
Matrigel coated, by BD (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Pd98059, by Merck Group (1 mentions)
S31 201, by Merck Group (1 mentions)
Sil 6r, by Thermo Fisher Scientific (1 mentions)
Propranolol hydrochloride, by Fujifilm (1 mentions)
Norepinephrine bitartrate salt, by Merck Group (1 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (1 mentions)
Mg132, by Fujifilm (1 mentions)
Anisomycin, by Fujifilm (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
21 protocols using sb203580
1
Modulating mEpiSCs Differentiation with AMPK Activators
2
Osteogenic Differentiation Assays
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SB203580 (p38 inhibitor), and a kit for enzyme activity staining of ALP were purchased from WAKO (Osaka, Japan). The Alizarin Red staining kit was obtained from PG Research (Tokyo, Japan). The Oil Red O stainning kit was purchased from Sigma-Aldorich (MO, USA). GsMTx4 and Yoda1 were purchased from Abcam (Tokyo, Japan) and Tocris Bioscience (Bristol, UK), respectively. Recombinant murine noggin was obtained from PeproTech (NJ, USA). U0126 was purchased from Cell Signaling (MA, USA).
3
Myogenic and Adipogenic Differentiation of Sorted Cells
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Sorted cells were cultured on Matrigel-coated (BD Biosciences) plates in GM at 37°C in 5% CO2 and 3% O2. Ten thousand cells were added per well. Myogenic and adipogenic differentiation were carried out as described in Supplemental Experimental Procedures . CD82 knockdown was performed by transfecting CD82 siRNA (Ambion). Cell proliferation was measured using Cell Counting Kit-8 (Dojindo). SB203580 (Wako), a p38 inhibitor, was used at 10 μM. APC (Haematologic Technologies) was used at 100 or 1,000 nM.
4
Norepinephrine Signaling Pathway Modulation
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We used (±)-norepinephrine (+)-bitartrate salt (Sigma-Aldrich, St Louis, MO), isoproterenol hydrochloride (Millipore, Billerica, MA), propranolol hydrochloride (Wako, Osaka, Japan), oxymetazoline hydrochloride (Wako), p38 inhibitor, SB203580 (Wako), MEK/ERK inhibitor, PD98059 (Millipore), STAT3 inhibitor, S31-201 (Sigma-Aldrich), recombinant human TGFβ1 (rTGFβ) (R&D systems, Minneapolis, MN), ET-1 (Sigma-Aldrich) and soluble IL-6 receptor (sIL-6R) (Peprotech, Rocky Hill, NJ).
5
Assessing HUVEC Senescence and Proliferation
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Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (Walkersville, MD, USA) and cultured according to the manufacturer's instructions. Endothelial cell proliferation was assessed by counting cell numbers after subculture. We defined senescent cells as those that did not increase in number and remained subconfluent after 2 weeks of culture. Senescence-associated β-galactosidase (SA-β-gal) staining was performed as described previously [14] (link). The number of population doublings (PD) was calculated as follows: PD = log (number of cells obtained/initial number of cells)/log 2. In some experiments, endothelial cells were treated with SB203580 (WAKO, Osaka, Japan, 10 µM), anisomycin (WAKO, 2 µM) or MG132 (WAKO, 0.5 µM).
6
Inhibition of JNK and p38 MAPK Signaling in AKAV Infection
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Antibodies specific for phospho-JNK1/2 (Thr183/Tyr185) (1:2000, #9251), JNK1/2 (1:2000, #9252), phospho-p38 MAPK (Thr180/Tyr182) (1:2000, #9211), p38 MAPK (1:2000, #9212), phospho-c-Jun (Ser63) (1:1000, #9261), phospho-HSP27 (Ser82) (1:1000, #2401), cleaved caspase-3 (Asp175) (1:1000, #9661), GAPDH (1:4000, #2118), and anti-rabbit IgG HRP-linked secondary antibody (1:10,000 for detection of phospho-c-Jun, phospho-HSP27, and cleaved caspase-3 and 1:20,000 for detection of the others, #7074) were purchased from Cell Signaling Technology (Danvers, USA).
The JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 were purchased from Wako (Osaka, Japan). We prepared 10 mM stocks of each inhibitor in dimethyl sulfoxide (DMSO). In the inhibition experiments, Vero E6 cells were infected with AKAV at an MOI of 0.5 for 1 h and then incubated in serum-free medium containing 20 μM of each inhibitor. Medium containing DMSO was used as the mock treatment control.
The JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 were purchased from Wako (Osaka, Japan). We prepared 10 mM stocks of each inhibitor in dimethyl sulfoxide (DMSO). In the inhibition experiments, Vero E6 cells were infected with AKAV at an MOI of 0.5 for 1 h and then incubated in serum-free medium containing 20 μM of each inhibitor. Medium containing DMSO was used as the mock treatment control.
7
Antibody and Reagent Sources for Cell Signaling
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An anti‐FLAG antibody (M2) was purchased from Merck Millipore (St. Charles, MO, USA). Anti‐cyclin A (SC‐751), anti‐cyclin D1 (SC‐753), anti‐CDC2 (SC‐163), anti‐CDK4 (SC‐601), anti‐H‐Ras (SC‐520), and anti‐β‐actin (SC‐130301) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). An anti‐phospho p65/RelA (S276) antibody was purchased from Rockland Immunochemicals (Limerick, PA, USA). Anti‐MSK1 and anti‐MSK2 antibodies were purchased from Cell Signaling Technology (#9309; Danvers, MA, USA) and BD Biosciences (San Jose, CA, USA), respectively. TNFα and PDGF were purchased from PeproTech (Rocky Hill, NJ, USA). LY294002, U0126, SP600125, and SB203580 were obtained from FUJIFILM Wako Pure Chemical (Osaka, Japan). NOX inhibitor, VAS2870, was purchased from Merck Millipore (Burlington, MA, USA).
8
Directed differentiation of HSPCs into T cells
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HSPCs were differentiated into T cells as previously described.14 (link) Briefly, thawed EB-derived HSPCs (from Ff-WJs513, Ff-WJs524, and Ff-WJs527 cells) were incubated in EB medium supplemented with 50 ng/mL VEGF-165A, 50 ng/mL rhbFGF, 50 ng/mL rhSCF, 50 ng/mL rhTPO, and 10 ng/mL rhFLT3L (day −2). The next day, cells were seeded into 48-well plates coated with RetroNectin (Takara Bio, 100 μg/mL) at a cell density of 2–4 × 104 cells/well. Titrated retrovirus was added to the cell suspension (day −1, MOI = 20). The next day, cells were seeded into plates coated with RetroNectin (5 μg/mL) and DLL4 protein (5 μg/mL) and incubated in alpha-MEM supplemented with 50 ng/mL rhSCF, 50 ng/mL rhIL-7 (FUJIFILM Wako), 50 ng/mL rhFLT3L, 100 ng/mL rhTPO, 30 nM rhSDF-1α (FUJIFILM Wako), 15 μM SB203580 (FUJIFILM Wako), 50 μg/mL PAA, 15% FBS, and 1% PSG (day 0). The medium was changed every 2–3 days, and cells were reseeded into fresh RetroNectin- and DLL4-coated 48-well plates every 7 days until day 21. Anti-CD3-BV510 (BioLegend) and anti-CD45-APC-Cy7 (BioLegend) were used for FACS analysis.
9
Intracellular Signaling Pathways Regulating RANTES Production
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Human recombinant GM-CSF was obtained from Tocris Bioscience, Bristol, UK. Substance P (Peptide Institute Inc., Osaka, Japan), SB203580 (Wako, Kanagawa, Japan), aprepitant (Cayman Chemical, Ann Arbor, Michigan), PD98059 (Wako), BIRB796 (Axon Medchem, Groningen, Netherlands), U0216 (Promega Corporation, Madison, WI) and mithramycin (Abcam, Cambridge, UK) were employed to investigate the intracellular signaling pathways involved in RANTES production. The actions of all these reagents are summarized in Table 1 .Table 1
Functional characteristics of chemical agents used.
| Chemical agents | Functions |
|---|---|
| Rottlerin | Protein kinase C inhibitor |
| TAPI-1 | Disintegrin and metalloproteinase inhibitor |
| PDTC | The radical scavenger |
| Perifosine | Akt inhibitor |
| U0126 | ERK1/2 inhibitor |
| Y-27632 | ROCK inhibitor |
| Dynasore | Dynamin inhibitor |
| aprepitant | Substance P/NK-1 receptor antagonists |
Akt: Protein kinase B; ROCK: Rho-associated coiled-coil forming kinase; NK-1: Neurokinin 1; ERK: Extracellular signal-regulated kinase.
10
Inhibition of Signaling Cascades
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FGF2 and AZD4547 (FGFR inhibitor) were purchased from R&D Systems, Inc. and Santa Cruz Biotechnology, Inc., respectively. SB203580 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) were purchased from FUJIFILM Wako Pure Chemical Corporation. U0126 [MAPK kinase (MEK) inhibitor], anti-ERK1/2 (cat. no. #9102), anti-phosphorylated (p)-ERK1/2 (cat. no. #9101), anti-p38 MAPK (cat. no. #8690), anti-p-p38 MAPK (cat. no. #9211), anti-stress-activated protein kinases (SAPK)/JNK (cat. no. #9252), anti-p-SAPK/JNK (cat. no. #4668) and anti-β-actin antibodies (cat. no. #4967) were purchased from Cell Signaling Technology, Inc.
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