Pd0325901

Manufactured by STEMCELL

PD0325901 is a small-molecule inhibitor of mitogen-activated protein kinase kinase (MEK) that selectively and potently inhibits the MEK1 and MEK2 enzymes. It is commonly used in cell culture and research applications.

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14 protocols using pd0325901

Detailed Cell Line Culture Protocols

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Human HeLa, HepG2, and HEK293T cell lines and mouse embryonic stem cells (mESC) and mouse embryonic fibroblasts (MEF) used in this study were all purchased from ATCC (the American Type Culture Collection). HeLa cell line was grown in DMEM (Gibco, 11965) media supplemented with 10% FBS and 1% 100X Pen/Strep (Gibco), while HepG2, HEK293T and MEF cell lines were maintained in DMEM (Gibco, 11995), supplemented with 10% FBS and 1% 100X Pen/Strep (Gibco). mESC cells were grown under a typical feeder-free culture condition: cells were cultured in DMEM (Gibco, 11995), supplemented with 15% FBS, 1% Pen/Strep (Gibco), 1X Glutamax (Gibco), 1X non-essential amino acids (Gibco), 1X 2-Mercaptoethanol (Gibco), and 1000 U/ml leukemia inhibitory factor (Millipore, ESG1107), together with two inhibitors: 3 μM CHIR99021 (STEMCELL Technologies, dissolved in DMSO) and 1 μM PD0325901 (STEMCELL Technologies, dissolved in DMSO). mESC cells were distinctively cultured on gelatinized culture plates (0.2% Gelatin). The culture of mESC cells were passaged every 2 days. All cells were cultured at 37 °C under 5.0% CO₂.

Zhang L.S., Liu C., Ma H., Dai Q., Sun H.L., Luo G., Zhang Z.S., Zhang L., Hu L., Dong X, & He C. (2019). Transcriptome-wide Mapping of Internal N7-methylguanosine Methylome in Mammalian Messenger RNA. Molecular cell, 74(6), 1304-1316.e8.

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Stem Cell Culture Protocols

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ESCs were grown on gelatin-coated plates in 2i media: 1:1 neurobasal and Dulbecco’s Modified Eagle’s Medium (DMEM)/F12, supplemented with 0.5× N2 (Thermo Scientific, #17502048), 0.5× B27 (Thermo Scientific, #17504044), 0.05% BSA (Thermo Scientific, #15260037), 1 mM PD0325901 (Stemcell Technologies, #72182), 3 μM CHIR99021 (Miltenyi Biotec, #130-103-926), 2 mM L-glutamine, 0.15 mM monothioglycerol, 100 U/ml LIF. NSCs were grown on laminin-coated (Sigma Aldrich, #L2020) plates in DMEM/F12 medium supplemented with 2 mM L-glutamine, 0.5× N2, B27, glucose, BSA, HEPES, and 10 ng/ml of both mouse EGF (Peprotech, #315-09) and human FGF-2 (Peprotech, #100-18B). MEFs were cultured in DMEM supplemented with 10% fetal bovine serum (FBS).

Haward F., Maslon M.M., Yeyati P.L., Bellora N., Hansen J.N., Aitken S., Lawson J., von Kriegsheim A., Wachten D., Mill P., Adams I.R, & Caceres J.F. (2021). Nucleo-cytoplasmic shuttling of splicing factor SRSF1 is required for development and cilia function. eLife, 10, e65104.

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Gastric Organoid Culture and Differentiation

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Single cell suspensions of mesenchymal cells and ENCCs were counted and appropriate volumes added to foregut spheroids collected in a 1.5mL tube at an approximate ratio of 1,000 ENCCs and 2,500 mesenchyme cells per spheroid. Cell mixtures were mixed via gentle pipetting, centrifuged at 300g for 3–5 minutes, gently resuspended into 50 μL of basement membrane Matrigel, and pipetted onto a TC dish to allow three-dimensional in vitro culture. Organoids were fed with a base media of Advanced DMEM/F12 supplemented with B27 supplement (1X), N2 supplement (1X), HEPES (13 mM), L-Glutamine (2 mM), penicillin-streptomycin (1X), and EGF (100 ng/mL). In addition to this base media, the first three days were supplemented with NOG (200 ng/mL) and RA (2 μM). In addition to EGF, hFGOs were supplemented with CHIR (2 μM) throughout the organoid outgrowth and also received a 48 hr. pulse of BMP4 (50 ng/mL) and PD0325901 (2 μM, Stem Cell Technologies) 96 hours prior to collection for parietal cell differentiation in vitro. Media was replaced every 3–4 days. Two weeks following spheroid embedding in Matrigel, the organoids were collected and re-plated in fresh Matrigel at a dilution of ~1:12.

Eicher A.K., Kechele D.O., Sundaram N., Berns H.M., Poling H.M., Haines L.E., Sanchez J.G., Kishimoto K., Krishnamurthy M., Han L., Zorn A.M., Helmrath M.A, & Wells J.M. (2021). Functional human gastrointestinal organoids can be engineered from three primary germ layers separately derived from pluripotent system cells. Cell stem cell, 29(1), 36-51.e6.

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Mouse E14Tg2A ESCs Maintenance and Modification

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Mouse E14Tg2A (E14) ESCs (male) were used for all experiments (Hooper et al. 1987 (link)) and to derive 2C-GFP reporter cells. 2C-GFP reporter ESCs were described previously (Percharde et al. 2018 (link)) and were used when prior purification of larger numbers of 2C-like cells was not needed or for validation. All ESCs were cultured at 37°C with 5% CO₂ on 0.1% gelatin-coated plates in ES-FBS culture medium (high-glucose DMEM GlutaMAX with sodium pyruvate [Thermo Fisher Scientific], 15% FBS [Gibco], 0.1 mM nonessential amino acids [Gibco], 0.1 mM 2-mercaptoethanol [Millipore], 1000 U/mL LIF supplement [ESGRO, Millipore]). Experiments comparing wild-type versus Dux^−/− E14 ESCs were performed in FBS/LIF media as above, supplemented with 2i (1 µM PD0325901 [Stem Cell Technologies], 3 µM CHIR99021 [Cambridge Bioscience]) as in Grow et al. (2021) (link). ESCs were routinely tested for mycoplasma and found to be negative. Inhibitors (Supplemental Table S2) were added to ESCs at the indicated concentrations and durations unless otherwise explicitly mentioned in the figure legends.

Xie S.Q., Leeke B.J., Whilding C., Wagner R.T., Garcia-Llagostera F., Low Y., Chammas P., Cheung N.T., Dormann D., McManus M.T, & Percharde M. (2022). Nucleolar-based Dux repression is essential for embryonic two-cell stage exit. Genes & Development, 36(5-6), 331-347.

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Feeder-free Culture of R1 ESCs

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R1 ESCs were cultured on 0.1% gelatin-coated plates without feeder cells in N2B27 medium (DMEM/F12 with 1:1 mix of GlutaMax/N2 and Neurobasal medium/B27, Invitrogen) supplemented with 2 mM L-Glutamine (Stemcell Technologies), 1x 2-Mercaptoethanol (Millipore), 1x NEAA (Stemcell Technologies), 3 μM CHIR99021 (Stemcell Technologies), 1 μM PD0325901 (Stemcell Technologies), 0.033% BSA solution (Invitrogen) and 10⁷ U/ml LIF (Millipore).

Avsec Ž., Weilert M., Shrikumar A., Krueger S., Alexandari A., Dalal K., Fropf R., McAnany C., Gagneur J., Kundaje A, & Zeitlinger J. (2021). Base-resolution models of transcription factor binding reveal soft motif syntax. Nature genetics, 53(3), 354-366.

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Optimizing Cell Differentiation Factors

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For functional experiments presented in Figs. 2f, 3f–i, 4c and Extended Data Figs. 6g,h,j and 10a,d, the following treatments were used: 10 µM SB431542 (STEMCELL Technologies), 1 µM XAV-939 (Sigma-Aldrich), 3 µM IWP-2 (Tocris), 10 µM PD0325901 (STEMCELL Technologies), 100 µM SU5402 (R&D systems), 1, 2 or 4 µM LDN (Sigma-Aldrich), 350 ng ml⁻¹ Noggin (R&D systems), 100 or 200 ng ml⁻¹ recombinant human BMP2 protein (Peprotech), 100, 200 or 400 ng ml⁻¹ BMP4 (Fisher Scientific), and 100 or 200 ng ml⁻¹ BMP7 (Peprotech). Subsequent medium changes were performed daily with the same concentration in the respective medium.

Pedroza M., Gassaloglu S.I., Dias N., Zhong L., Hou T.C., Kretzmer H., Smith Z.D, & Sozen B. (2023). Self-patterning of human stem cells into post-implantation lineages. Nature, 622(7983), 574-583.

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Mouse Embryonic Stem Cell Culture

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We cultured mESCs in DMEM (Invitrogen) supplemented with 15% FBS (GeminiBio), 1% nucleosides (100×) (Millipore), 1 mmol/L l-glutamine (Gibco), 1% nonessential amino acid (Gibco), 0.1 mmol/L 2-mercaptoethanol (Sigma), 1,000 U/mL LIF (Millipore), 3 μmol/L CHIR99021 (Stemcell), and 1 μmol/L PD0325901 (Stemcell) in 37°C and 5% CO₂.

Dou X., Huang L., Xiao Y., Liu C., Li Y., Zhang X., Yu L., Zhao R., Yang L., Chen C., Yu X., Gao B., Qi M., Gao Y., Shen B., Sun S., He C, & Liu J. (2023). METTL14 is a chromatin regulator independent of its RNA N6-methyladenosine methyltransferase activity. Protein & Cell, 14(9), 683-697.

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Culturing Diverse Cell Lines for Research

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HEK293T, A549 and MCF-7 cells were purchased from the American Type Culture Collection. mES cells were obtained as a kind gift from J. Hanna’s laboratory. HEK293T and MCF-7 cells were maintained in DMEM (11995065, Gibco) supplemented with 10% FBS and 100 U penicillin–streptomycin (15140148, Gibco). A549 cells were grown in Ham’s F-12K medium (21127022, Gibco) supplemented with 10% FBS and 100 U penicillin–streptomycin. mES cells were cultured in Knockout DMEM (10829018, Gibco) supplemented with 15% heat-inactivated FBS, 100 U penicillin–streptomycin, 1× GlutaMAX (35050061, Gibco), 55 µM β-mercaptoethanol, 1× MEM non-essential amino acid solution (11140076, Gibco), 10³ U ml⁻¹ LIF (ESG1107, ESGRO), 3 µM CHIR99021 (72052, STEMCELL Technologies) and 1 µM PD0325901 (72182, STEMCELL Technologies). All cell types were grown in sterile cell culture incubators at 37 °C and 5% CO₂. Cell lines were not authenticated. All cell types tested negative for mycoplasma contamination. Mycoplasma contamination was routinely tested with Hoechst staining.

Despic V, & Jaffrey S.R. (2023). mRNA ageing shapes the Cap2 methylome in mammalian mRNA. Nature, 614(7947), 358-366.

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Transcriptional Profiling of Stem Cells

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E14Tg2a.4 mouse embryonic stem cells (ECACC General Cell Collection; catalogue number: 08021401) were cultured on 0.1% gelatin-coated culture plates in DMEM (4,5 g/l glucose) supplemented with GLUTAMAX-I, 15% heat-inactivated fetal calf serum (42F5874K, ESC culture tested, GIBCO), 0.1 mM beta-mercaptoethanol, 0.1 mM nonessential amino acids 1500 U/ml leukemia inhibitory factor (produced in house), 3 µM CHIR99021 (72054, Stem Cell Technologies) and 1 µM PD0325901 (72184, Stem Cell Technologies) in 5% CO₂ at 37 °C.
IMR90 primary human fetal lung fibroblast cells (CORIELL Institute for Medical Research, Reference: I90-19) were cultured in DMEM 41966 (4,5 g/l glucose) supplemented with 10% fetal calf serum, Penicillin 100 UI/ml, and Streptomycin 100 µg/ml in 5% CO2 at 37 °C. The cells were at passage 21 when performing the experiments. For both scRNAseq and FACS experiments, 20,000 cells per well were seeded into 6 well plates and cultured for 72 h.

Riba A., Oravecz A., Durik M., Jiménez S., Alunni V., Cerciat M., Jung M., Keime C., Keyes W.M, & Molina N. (2022). Cell cycle gene regulation dynamics revealed by RNA velocity and deep-learning. Nature Communications, 13, 2865.

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Generation of Mettl3 Knockout mESCs

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Human HeLa cells and mouse embryonic stem cells (mESC) were purchased from ATCC. HeLa cells were grown in DMEM (Gibco, 11965092) media supplemented with 10% FBS (Gibco) and 1% 100× Pen/Strep (Gibco). WT, control knockout, and Mettl3 conditional knockout (cKO) mESCs were maintained in DMEM (Invitrogen) supplemented with 15% FBS (Gibco), 1% nucleosides (100×) (Millipore), 1 mM L-glutamine (Gibco), 1% nonessential amino acids (Gibco), 0.1 mM 2-mercaptoethanol (Sigma), 1,000 U/ml LIF (Millipore), 3 μM CHIR99021 (Stemcell), and 1 μM PD0325901 (Stemcell). All cells were cultured at 37 °C under 5.0% CO₂.
Mettl3 cKO mES cell lines were generated following previously reported methods⁵³. Briefly, mESCs derived from Mettl3^flox/flox mouse blastocyst were transfected with 200 ng PB-CAG-Puromycin-P2A-CreERT2 and 100 ng PBase by electroporation. After 24 h, electroporated cells were treated with 1 μg/ml Puromycin to generate stable Mettl3^flox/flox; CreERT2 mES clones. To induce deletion, Mettl3^flox/flox; CreERT2 ESC cells were treated with 1 μg/ml 4-hydroxytamoxifen (Sigma). These Mettl3 KO cells were cultured for 48 h before harvesting. Untreated Mettl3^flox/flox; CreERT2 ESC cells were used as ctrl mESCs.

Stampfer M.R., Bodnar A., Garbe J., Wong M., Pan A., Villeponteau B, & Yaswen P. (1997). Gradual Phenotypic Conversion Associated with Immortalization of Cultured Human Mammary Epithelial Cells. Molecular Biology of the Cell, 8(12), 2391-2405.

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