Mek inhibitor pd0325901

Manufactured by Selleck Chemicals

Sourced in United States

PD0325901 is a MEK inhibitor, a class of compounds that selectively target the mitogen-activated protein kinase (MAPK) pathway. It acts by inhibiting the enzymatic activity of MEK, a key component of the MAPK signaling cascade. PD0325901 has been widely used in research applications to study the role of the MAPK pathway in various cellular processes.

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10 protocols using mek inhibitor pd0325901

Maintenance of Mouse Embryonic Stem Cells

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Mouse embryonic stem cell line E14TG2a (mES cells) was cultured with the N2B27 base medium supplemented with 1 mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.15 mM 1-thioglycerol (Sigma), 100 U/ml of penicillin–streptomycin (Invitrogen), 25 μg/ml of BSA (Sigma), 1 μM MEK inhibitor PD0325901 (Selleck Chemicals), 3 μM GSK3β inhibitor CHIR99021 (Selleck Chemicals), 2% KOSR (Thermo Fisher), and 1000 U/ml of ESGRO leukemia inhibitory factor LIF (Millipore) on plates coated with 0.2% gelatin.

Xiao Z., Liu S., Li Z., Cui J., Wang H., Wang Z., Ren Q., Xia L., Wang Z, & Li Y. (2022). The Maternal Microbiome Programs the m6A Epitranscriptome of the Mouse Fetal Brain and Intestine. Frontiers in Cell and Developmental Biology, 10, 882994.

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Serum-free culture of E14Tg2a stem cells

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E14Tg2a (129/Ola isogenic background) and the derived cell lines were cultured on 0.1% gelatin-coated plates in serum-free DMEM/F12 (Gibco) and Neurobasal (Gibco) medium (1:1) supplemented with N-2 (Gibco), B-27 (Gibco), BSA (0.05%, Gibco), 10⁴ U of Leukemia Inhibitory Factor/LIF (Millipore), MEK inhibitor PD0325901 (1 μM, Selleckchem), GSK3-β inhibitor CHIR99021 (3 μM, Cayman Chemical) and 1-Thioglycerol (1.5 × 10^-4 M, Sigma-Aldrich). The cell lines were passaged every 2 days in daily culture. During the protein depletion experiments, the cells were seeded overnight before the start of the time course in the following densities: For a 96 h time course, 2.5 k, 35 k, 150 k, and 400 k cells were seeded in 24-well, 6-well, 10-cm and 15-cm plates, respectively. For 24 h time course, 5 k, 0.5 M, and 4 M cells were seeded in chamber slide (ThermoFisher Scientific), 6-well and 15-cm plates, respectively. The media were refreshed or the cells were split in 1:10 every 2 days during a time course.

Liu N.Q., Maresca M., van den Brand T., Braccioli L., Schijns M.M., Teunissen H., Bruneau B.G., Nora E.P, & de Wit E. (2020). WAPL maintains a cohesin loading cycle to preserve cell-type specific distal gene regulation. Nature genetics, 53(1), 100-109.

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Isolation and Culture of Mouse HSPCs

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Primary hematopoietic stem and progenitor cells (HSPCs) were obtained as described in (28 (link)). Briefly, cKit magnetic beads (Miltenyi Biotec) were used to enrich HSPCs from mouse bone marrow and cultured in IMDM (Gibco) supplemented with 15% FBS (Gibco), 10 ng/ml mIL-6, 10 ng/ml mIL-3, 50 ng/ml SCF, 20 ng/ml thrombopoietin (PeproTech) and 10 ng/ml Flt3 ligand (PeproTech).
The E14 and K562 cell lines were obtained from the experimental pathology cell bank in State Key Laboratory of Experimental Hematology. The K562 cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS. E14 cells were maintained in 0.2% gelatin (Sigma)-coated plates in 2i medium, which consisted of DMEM/F12 supplemented with Neurobasal medium, serum-free N2B27 medium supplemented with 10 μM MEK inhibitor PD0325901 and 30 μM GSK3 inhibitor CHIR99021 (both from Selleckchem), 2% KnockOut™ serum replacement (Gibco), 0.002% BSA (Gibco), 1 mM MTG (Gibco), 1000 U/ml LIF (Millipore), 0.1 mM non-essential amino acid and 2 mM GlutaMAX Supplement (Gibco).

Zhang H., Lu T., Liu S., Yang J., Sun G., Cheng T., Xu J., Chen F, & Yen K. (2021). Comprehensive understanding of Tn5 insertion preference improves transcription regulatory element identification. NAR Genomics and Bioinformatics, 3(4), lqab094.

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Culturing Mouse Embryonic Stem Cells

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F1 hybrid 129/Castaneus female mouse embryonic stem cells (gift from Kathrin Plath) were cultured in serum-free N2B27-based medium (250 ml Neurobasal media (Gibco), 250 ml DMEM/F12 (Gibco), 5 ml 100× N2 supplement (Gibco), 5 ml 50× B27 supplement (Gibco), 5 ml 200 mM L-Glutamine (Gibco), 3.6 μl 2-mercaptoethanol, 50 μg human leukemia initiation factor (5 × 10⁵ units, EMD Millipore), 7.4 μg Progesterone, 10 mg Bovine Insulin (Sigma), 350 μl 7.5% BSA Fraction V (Gibco), supplemented with MEK inhibitor PD0325901 (50 μl 10 mM, SelleckChem), and GSK3b inhibitor CHIR99021 (150 μl 10 mM, SelleckChem)). Prior to plating cells, tissue culture dishes were pretreated with PBS + 0.2% gelatin (Sigma) and 1.75 μg/ml laminin (Sigma) for 2–10 hours at 37°C. At each passage, cells were trypsinized for 3–5 minutes in TVP Solution (0.025% trypsin, 1% Chicken Serum (Sigma), and 1 mM EDTA in PBS pH 7.4) at room temperature. Cells tested negative for mycoplasma contamination and were authenticated by comparing polymorphisms to 129S1 and Castaneus genomes.

Engreitz J.M., Haines J.E., Perez E.M., Munson G., Chen J., Kane M., McDonel P.E., Guttman M, & Lander E.S. (2016). Local regulation of gene expression by lncRNA promoters, transcription, and splicing. Nature, 539(7629), 452-455.

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EGF Signaling Pathway Modulation

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Cells were serum starved for 24 hours and stimulated with 20 ng/mL EGF (Corning) in serum free media for 4 or 24 hours before subsequent lysis and analysis by quantitative PCR or Western, respectively. For inhibitor studies, cells were pre-incubated with 0.25 μM MEK inhibitor PD0325901 (Selleckchem) or 0.25 μM PI3K inhibitor LY294002 (Sigma) for 30 minutes before the addition of EGF. Cells were treated with 10 μM DNMT inhibitor 5-aza-2-deoxycytidine (Sigma) and 10 μM EZH2 inhibitor GSK343 (Selleckchem) in complete media for 72 hours.

Lerman I., Ma X., Seger C., Maolake A., Garcia-Hernandez M.D., Rangel-Moreno J., Ackerman J., Nastiuk K.L., Susiarjo M, & Hammes S.R. (2019). Epigenetic Suppression of SERPINB1 Promotes Inflammation-Mediated Prostate Cancer Progression. Molecular cancer research : MCR, 17(4), 845-859.

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Serum-free culture of E14Tg2a stem cells

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MTT Cytotoxicity Assay Protocol

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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), N-acetyl-L-cysteine (NAC) and crystal violet were purchased from Sigma-Aldrich (St. Louis, MO). DMEM, fetal bovine serum (FBS), and the antibiotic mixture (penicillin-streptomycin) were from Invitrogen (Carlsbad, CA, USA). PLX4032 and MEK inhibitor (PD0325901) were purchased from Selleck Chemical (Houston, TX). All other solvents or chemicals were of reagent or HPLC grade.

Feng J.H., Nakagawa-Goto K., Lee K.H, & Shyur L.F. (2016). A novel plant sesquiterpene lactone derivative DETD-35 suppresses BRAFV600E mutant melanoma growth and overcomes acquired vemurafenib resistance in mice. Molecular cancer therapeutics, 15(6), 1163-1176.

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Bovine Somatic Cell Reprogramming

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The DOT1L inhibitor EPZ004777 (iDOT1L) was purchased from AOBIOUS Inc. (Gloucester, MA, USA). WNT inhibitor IWR1, GSK-3 inhibitor CHIR-99021, MEK inhibitor PD0325901, ROCK inhibitor Y-27632 (ROCKi), and caspase inhibitor Z-VAD-FMK were purchased from Selleckchem (Houston, TX, USA). Forskolin was purchased from Fisher Scientific (Pittsburg, PA, USA). Human reprogramming factors OCT4, SOX2, KLF4, MYC, NANOG, LIN28A, and KDM4A were used for bovine somatic cell reprogramming. The constructs pMXs-OCT4, FUW-TetO-eGFP, and FUW-m2rtTA were purchased from Addgene (Cambridge, MA, USA). Construction of the polycistronic vector pMXs-KLF4, MYC, and SOX2 (KMS) [68 (link)] and pMXs-LIN28A and NANOG (LN) [30 (link)] were described previously. Human KDM4A was cloned into pMXs-vector similarly to as previously described in [30 (link)]. Primary bovine umbilical cord-derived mesenchymal stem cells (bMSCs) were collected from Wharton’s jelly part of the placenta of a newborn male Holstein calf, and were maintained with low serum MSC medium (ATCC, Manassas, VA, USA).

Su Y., Wang L., Fan Z., Liu Y., Zhu J., Kaback D., Oudiz J., Patrick T., Yee S.P., Tian X.(., Polejaeva I, & Tang Y. (2021). Establishment of Bovine-Induced Pluripotent Stem Cells. International Journal of Molecular Sciences, 22(19), 10489.

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Islet Proliferation Assay with PL

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In proliferation experiments mouse islets were cultured in RPMI supplemented with Glutamax, penicillin (100 IU/ml), streptomycin (100 IU/ml) and 10% fetal calf serum either with or without PL 500 ng/ml (Affiland, Liège, Belgium) for 96 h. BrdU 30 μg/ml (Sigma-Aldrich) was added for the last 48 h. For RNA isolation the islets were cultured in RPMI supplemented with Glutamax, penicillin (100 IU/ml), streptomycin (100 IU/ml), 10% fetal calf serum and stimulated with 500 ng/ml PL (Affiland), 2 μM gefitinib (Selleckchem, Houston, TX, USA), 10 nM rapamycin (Selleckchem) or 0,5 μM MEK inhibitor PD0325901 (Selleckchem) for 96 h. All inhibitors were dissolved in DMSO and DMSO 0,02% was also added to the control and PL only media. After stimulation the islets were lysed in the RNA extraction buffer (NucleoSpin RNAII kit).

Hakonen E., Ustinov J., Palgi J., Miettinen P.J, & Otonkoski T. (2014). EGFR Signaling Promotes β-Cell Proliferation and Survivin Expression during Pregnancy. PLoS ONE, 9(4), e93651.

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Culturing Mouse Embryonic Stem Cells

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Mouse ES cells were cultured as described in Koehler and Hashino 47 . Briefly, cells were maintained on dishes coated with 0.1% gelatin (Millipore) in ESC culture medium, a serum-free 2i+LIF medium containing 47.75% Advanced DMEM/F-12 (Gibco), 47.75% Neurobasal (Gibco), 0.5% N-2 supplement (Gibco), 1% B-27 supplement minus Vitamin A (Gibco), 1% GlutaMax (Gibco), 1% Penicillin-Streptomycin (Corning), 1%
EmbryoMax 2-mercaptoethanol (MilliporeSigma), and supplemented with 1 µM MEK inhibitor PD0325901 (Selleckchem), 3 µM GSK3 inhibitor CHIR99021 HCl (Selleckchem) and 1,000 units/mL mouse LIF protein (MilliporeSigma).
For Alkaline Phosphatase (AP) staining, 4 x 10 3 single cells were seeded into one well of a 24-well plate. 4 days later, AP staining was done using Alkaline Phosphatase Detection Kit (MilliporeSigma)
following the manufacturer's instructions.

Xie G., Lee J., Senft A.D., Park Y., Chakraborty S., Thompson J.J., Liu C., Macfarlan T.S., Rocha P.P., Peng W., & Ge K. (2020). MLL3/MLL4 methyltransferase activities control early embryonic development and embryonic stem cell differentiation in a lineage-selective manner.

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