B7 and H12 were seeded in 6-well plates in DMEM containing 10% FBS. After the cells reached 70% confluence, they were first treated with the selective MEK1/2 inhibitor U0126 (3, 10, and 30 µM; Wako, Osaka, Japan) or the MEK1 inhibitor PD98059 (30, 60, and 90 µM; Wako) in DMEM containing 10% FBS. Cell numbers were counted at 12, 24, and 48 h. For RT-PCR, the cells were treated with U0126 (10 and 30 µM) or PD98059 (90 µM) and were harvested at 24 and 48 h.
Pd98059
Manufactured by Fujifilm
Sourced in Japan, United States
PD98059 is a laboratory chemical compound used in cell biology research. It is a specific inhibitor of the mitogen-activated protein kinase kinase (MEK) enzyme, which plays a role in the regulation of cellular processes. The core function of PD98059 is to inhibit the activation of the MEK enzyme, allowing researchers to study the effects of MEK inhibition on cellular signaling pathways and related biological processes.
Automatically generated - may contain errors
Lab products found in correlation
U0126, by Fujifilm (3 mentions)
Sb203580, by Fujifilm (3 mentions)
Pd0325901, by Fujifilm (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Recombinant mouse egf, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 1β, by Miltenyi Biotec (1 mentions)
Recombinant human fgf1, by R&D Systems (1 mentions)
Bay 11 7085, by Cayman Chemical (1 mentions)
Tpca 1, by R&D Systems (1 mentions)
Su5402, by Fujifilm (1 mentions)
Bay 11 7085, by Fujifilm (1 mentions)
Heparin sodium salt, by Merck Group (1 mentions)
Tnf α, by Miltenyi Biotec (1 mentions)
Forskolin, by Fujifilm (1 mentions)
Chir99021, by Fujifilm (1 mentions)
N2b27 medium, by Thermo Fisher Scientific (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
A83 01, by Fujifilm (1 mentions)
14 protocols using pd98059
1
MEK Inhibitors Influence Cell Growth
2
Modulation of Myofibroblast Differentiation
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Recombinant mouse EGF was purchased from PeproTech, Inc. Recombinant human IL-1β and TNF-α were obtained from Miltenyi Biotec, GmbH (Bergisch Gladbach). The MEK inhibitors U0126 and PD98059, and the EGFR inhibitor PD153035 were purchased from Calbiochem (Merck KGaA). The NF-κB inhibitor BAY 11-7085 was obtained from Cayman Chemical. Recombinant human FGF-1 and the NF-κB kinase-2 (IKK-2) inhibitor TPCA-1 were purchased from R&D Systems, Inc. The FGFR1 inhibitor SU-5402 was obtained from Wako Pure Chemical Industries, Ltd. We confirmed that dimethyl sulfoxide (DMSO), the vehicle used for the U0126, PD98059, PD153035, BAY 11-7085, TPCA-1, and SU-5402 treatments, did not affect the expression of the MF markers α-SMA and type I collagen (data not shown). Heparin sodium salt was obtained from Merck KGaA. Heparin was included to achieve the optimal FGF-1 activity (28 (link)).
3
Differentiation of iPSCs into Neural Stem Cells
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The HDDPC-derived iPSCs were cultured in NSC medium, based on N2B27 medium containing DMEM/F12 (#12634-010; Thermo Fisher Scientific, Inc., Waltham, MA, USA), Neurobasal medium (#21103-049; Thermo Fisher Scientific, Inc.), N2 supplement (#17502-048; Invitrogen, Carlsbad, CA, USA), and B27 supplement (#17504044; Invitrogen) in a ratio of 48:48:1:2, respectively. In the NSC medium, 5 ng/mL recombinant human bFGF, 0.02 μg/mL recombinant human LIF, 1 μM PD0325901 (#162-25291; Wako Pure Chemical Industries, Ltd.), 10 μM PD98059 (#169-19211; Wako Pure Chemical Industries, Ltd.), 3 μM CHIR99021 (#038-23101; Wako Pure Chemical Industries, Ltd.), 10 μM forskolin (#067-02191; Wako Pure Chemical Industries, Ltd.), and 1 μM kenpaullon (#110-00831; Wako Pure Chemical Industries, Ltd.) were also included. Medium change and cell passage were done as for cultivation of EpiSCs, as mentioned above.
4
Differentiation of Human iPSC-Derived Enterocytes
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The enterocytes were differentiated as reported [27 (link)]. Briefly, human iPSC-derived small intestinal stem cells were subcultured in 24- or 96-well plates or 24-well inserts and cultured using enterocyte maturation medium and 10 μM Y−27632 (Focus Biomolecules) for 24 h. The enterocyte maturation medium consisted of DMEM/F12 containing 2% FBS, 2× B27 supplement, 1× N2 supplement, 1% NEAA, 100 units/mL penicillin G, 100 µg/mL streptomycin sulfate, 1% GlutaMax, and 50 ng/mL epidermal growth factor (PeproTech Inc.). Then, cells were cultured in enterocyte maturation media supplemented with 30 μM forskolin for 18 days. From the seventh day of the 18-days culture, PD98059 (Wako Pure Chemical Industries), 5 µM 5-aza−2′-deoxycytidine (5-aza−2′-dC) (Wako Pure Chemical Industries), and 0.5 µM A−83–01 (Wako Pure Chemical Industries) were added to mature the human iPSC-derived enterocyte (HiEnt).
5
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.
6
Small Molecule Inhibitors Reconstitution
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The four small molecule inhibitors (YPPP) were first reconstituted into stock solutions; 10 mM Y27632 (Fujifilm Wako) was prepared in sterilized phosphate buffered saline (PBS), and 40 mM PD0325901 (Fujifilm Wako), 10 mM PD173074 (Fujifilm Wako) and 10 mM PD98059 (Fujifilm Wako) were prepared in dimethyl sulfoxide (DMSO, Nakarai tesque). Stock solutions were stored at −20°C until use. Y27632, PD0325901, PD173074 and PD98059 were used at final concentrations of 50, 0.04, 0.01, 6.3 μM.
7
Preparation of Cell Culture Media
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Recombinant TGFβ (Wako Pure Chemical, Tokyo, Japan) and recombinant PDGF-BB (Wako Pure Chemical) were dissolved in Milli Q water. Tranilast (Tokyo Kasei Kogyo Co., Tokyo, Japan), imatinib (Phoenix Pharmaceuticals, Belmont, CA, USA), pirfenidone (Tokyo Kasei Kogyo Co.), cromoglicate (Tokyo Kasei Kogyo Co.), bosutinib (KareBay Biochem, Ningbo, China), dasatinib (Cayman Chemical, Ann Arbor, MI, USA), CP673451 (AdooQ BioScience, Irvine, CA, USA), LY294002 (LC Laboratories, Woburn, MA, USA), PD98059 (Wako Pure Chemical), and PP2 (Cayman Chemical) were dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in each cell culture did not exceed 0.5% (v/v).
8
IGFBP-3 Regulation of IGF-IR, MEK, and AKT
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Following siRNA treatment, fresh culture media were replaced and cells were treated with specific inhibitors of either IGF-IR (NVP-AEW541, 5 μM for 30 min; Novartis Pharmaceuticals, Basel, Switzerland), MEK (PD98059, 20 μM for 90 min; Wako Pure Chemical Industries, Osaka, Japan), or AKT (MK-2206, 5 μM for 30 min; Cayman Chemical, Ann Arbor, MI). Cells were also treated with 100 ng/mL recombinant human IGFBP-3 from either of two sources for either 90 min or 24 h before further experimental manipulation; the first was #675-B3, derived from NS0 mouse myeloma cell line (R&D Systems, Minneapolis, MN) and the second was ab280941, derived from HEK293T, a human embryonic kidney cell line (Abcam, Cambridge, UK).
Recombinant human IGFBP-3 proteins were reconstituted in a solution of 0.1% bovine serum albumin (BSA) in sterile PBS prior to use.
Recombinant human IGFBP-3 proteins were reconstituted in a solution of 0.1% bovine serum albumin (BSA) in sterile PBS prior to use.
9
Measurement of Renal NBCe1 Activity
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NBCe1 activity was determined as previously described (18 (link), 22 (link), 62 (link)). Briefly, the PT (S2 segment) fragment was manually microdissected from rat or human kidneys without collagenase treatment, and it was transferred to a perfusion chamber mounted on an inverted microscope. To avoid the influence of luminal transporters, the PT fragment was collapsed with two holding pipettes. The luminally collapsed PT was incubated with an acetoxymethyl ester form of a pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (Dojindo Laboratories) in Dulbecco’s modified Eagle’s medium (DMEM) for 10 min, and pHi was monitored with a photometry system, MetaFluor 7.7 software (Molecular Devices). The chamber was perfused with prewarmed (38 °C) DMEM equilibrated with 5% CO2/95% O2 gas, and subsequently, bath HCO3− concentrations were repeatedly switched from 25 to 12.5 mM in the absence and presence of azPC (Cayman Chemical Company) or other chemical agents such as a specific PPARγ antagonist GW9662 (Sigma–Aldrich) at 5 μM and an MEK inhibitor PD98059 (FUJIFILM Wako Pure Chemical) at 10 μM, both of which exhibit sufficient inhibitory activities without affecting the basal NBCe1 activity in PTs (19 (link), 22 (link)). NBCe1 activity was calculated using the rate of pHi decrease in response to bath HCO3− reduction and buffer capacity.
10
Modulation of Inflammatory Signaling Pathways
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Recombinant human IL-33 was purchased from Pepro Tech (Rocky Hill, NJ, USA). The mouse monoclonal anti-human MCP-1 antibody was from Santa Cruz Biotechnology (Heidelberg, Germany). The rabbit polyclonal antibodies for JNK, phospho-JNK (Thr183/Tyr185), p38, phospho-p38 (Thr180/Tyr182), ERK1/2, phospho-ERK1/2 (p42/44 MAPK), c-Jun, and phospho-c-Jun (Ser73) were obtained from Cell Signaling Technology (Beverly, MA, USA). SP600125 (JNK inhibitor) was purchased from BIOMOL (Plymouth Meeting, PA, USA). Simvastatin, PD98059 (ERK1/2 inhibitor), and SB203580 (p38 MAPK inhibitor) were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Mevalonate was obtained from Sigma (St Louis, MO, USA).
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