A volume of 100 μL per well of cells in supplemented media were incubated at 37°C and 5% CO2 with doxorubicin (Sigma-Aldrich, Milwaukee, WI, USA), idarubicin (Sigma-Aldrich), BEZ-235 (provided by Novartis, Inc.), or selumetinib (ChemieTek, Indianapolis, IN, USA). Each compound was plated using a nine-point logarithmic concentration scale ranging from 15 nM to 100 μM. Stock solutions of doxorubicin and idarubicin (10 mM) were prepared in water while stock solutions of BEZ-235 and selumetinib (25 mM and 75 mM, respectively) were prepared in 100% dimethyl sulfoxide (DMSO) (Sigma-Aldrich). Subsequent dilutions and controls were prepared to account for the inclusion of water or DMSO in the stock solution.
Selumetinib
Manufactured by Merck Group
Sourced in United States
Selumetinib is a small molecule inhibitor developed by the Merck Group. It is designed to target and inhibit the activity of the MEK1 and MEK2 enzymes, which are part of the MAPK/ERK signaling pathway. Selumetinib can be used as a research tool to study the role of the MAPK/ERK pathway in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Selumetinib, by Selleck Chemicals (2 mentions)
Benzoxathiole, by Abcam (2 mentions)
Easysep pe positive selection kit, by STEMCELL (2 mentions)
Ly294002, by Merck Group (2 mentions)
Cyclosporin a, by Merck Group (2 mentions)
Dead cell removal kit, by Miltenyi Biotec (2 mentions)
Idarubicin, by Merck Group (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Selumetinib, by Chemietek (1 mentions)
Bez235, by Merck Group (1 mentions)
Bez235, by Novartis (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
6 well plate, by Corning (1 mentions)
Trametinib, by Merck Group (1 mentions)
P1754, by Merck Group (1 mentions)
Ftp 20 38 50, by Instech (1 mentions)
H7509, by Merck Group (1 mentions)
Atazanavir, by Merck Group (1 mentions)
Ritonavir, by Merck Group (1 mentions)
9 protocols using selumetinib
1
Cytotoxicity Assay of Chemotherapeutics
2
Acrolein and Selumetinib Dissolution Protocol
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The chemicals used were acrolein (Sigma, St. Louis, MO, USA) and selumetinib (Abmole Bioscience, Houston, USA). selumetinib was dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA) and was diluted with DMEM or Neurobasal (NB, Thermo Fisher Scientific, Waltham, MA, USA) medium.
3
Selumetinib Inhibits GBM Cell Growth
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Selumetinib (Selleck Chemicals, Houston, TX, USA) was used to inhibit MEK1/2 phosphorylation. GBM cells were treated with 0.195, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 µmol/L Selumetinib dissolved in dimethyl sulfoxide (DMSO, Sigma‐Aldrich, St. Louis, MO, USA) for 72 h.
Lentiviruses containing an EGFL7 inhibitor sequence (shEGFL7) or negative control sequences were purchased from GeneChem (Shanghai, China) (Supplementary Table S1). The transfection process follows GeneChem's instructions. U87‐MG and U251‐MG cells were infected with lentiviruses at 70% confluence. The infected cells (named U87‐EGFL7kd and U251‐EGFL7kd) were harvested for further use after 48 h. A universal negative control lentiviral vector was used as control.
Lentiviruses containing an EGFL7 inhibitor sequence (shEGFL7) or negative control sequences were purchased from GeneChem (Shanghai, China) (Supplementary Table S1). The transfection process follows GeneChem's instructions. U87‐MG and U251‐MG cells were infected with lentiviruses at 70% confluence. The infected cells (named U87‐EGFL7kd and U251‐EGFL7kd) were harvested for further use after 48 h. A universal negative control lentiviral vector was used as control.
4
Selumetinib Modulates miR-302a in HCC1937 and MDA-MB-231 Cells
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After planted in 96- or 6-well plates (Corning, USA) using DMEM with 10 % fetal bovine serum (FBS) at 37 °C in humidified 5 % CO2, HCC1937 and MDA-MB-231 cells were exposed for 24 h to various doses of Selumetinib(Sigma-Aldrich, Louis, MO). For the transfected process, cells were with starved in DMEM without FBS for 6 h, then miR-302a-AMO, miR-302a-MIMIC, NC or pcDNA3.1-CUL1 were added with Lipofectamine 2000 Reagent (Invitrogen) following the manufacturer’s protocol. Cell proliferation assays were performed with tetrazolium salt (MTT) array according to the manufacturer’s protocol.
5
Selumetinib and Trametinib Oral Gavage
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Selumetinib (AZD6244, S1008) was purchased from Selleckchem and dissolved in DMSO at stock dilutions of 30 mg/mL under sterile conditions.
Working dilutions of Trametinib and Selumetinib were dissolved at a dose volume of 0.2 mL/20 g body weight in 0.5% hydroxypropylmethylcellulose (Sigma-Aldrich H7509), 0.2% Tween-80 (Sigma-Aldrich P1754) in distilled water (pH 8.0). Adult mice were treated at specific concentrations of Trametinib or Selumetinib by oral gavage (plastic feeding tubes, 20ga x 38 mm, Instech, FTP-20-38-50), every day for 14 days, and then sacrificed at 4 h post dose. Control mice were treated with vehicle only (DMSO).
Working dilutions of Trametinib and Selumetinib were dissolved at a dose volume of 0.2 mL/20 g body weight in 0.5% hydroxypropylmethylcellulose (Sigma-Aldrich H7509), 0.2% Tween-80 (Sigma-Aldrich P1754) in distilled water (pH 8.0). Adult mice were treated at specific concentrations of Trametinib or Selumetinib by oral gavage (plastic feeding tubes, 20ga x 38 mm, Instech, FTP-20-38-50), every day for 14 days, and then sacrificed at 4 h post dose. Control mice were treated with vehicle only (DMSO).
6
Screening Pharmaceutical Inhibitors
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Ritonavir (cat# SML0491), atazanavir (cat# SML1796), lopinavir (cat# SML1222), sorafenib (cat# SML2653), and selumetinib (cat# AMBH2D6F1825) were from Sigma-Aldrich. Verteporfin (cat# 5305) was from Tocris Bioscience and Bio-Techne, and VT107 from Vivace Therapeutics.
7
Evaluating Targeted Therapies in ATC Cells
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Two ATC cell lines, 8505C and Cal-62 (kindly provided by Prof. James Fagin, Memorial Sloan Kettering Cancer Center), harboring a BRAF V600E or KRAS G12R mutation, respectively, were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum, 1 mM pyruvate, and gentamycin. Before use, the cell lines were authenticated by short tandem repeat profiling (17) . The cells were incubated with dimethylsulfoxide (vehicle control); with the DLCs digoxin, strophanthin K, lanatoside C, digoxigenin (all from Sigma-Aldrich), or proscillaridin A (Santa Cruz Biotechnology); or with the MAPK/ERK-inhibitor selumetinib, the BRAF V600E inhibitor dabrafenib, or the mammalian-target-of-rapamycin inhibitor rapamycin (all from Sigma-Aldrich) for indicated time points and concentrations. For all compounds, stock concentrations ranged between 50 and 100 mM in dimethylsulfoxide vehicle. Compound selection and applied concentrations were based on earlier studies (7, 8, 18, 19) .
8
Tcf7ΔGzmb CD8+ Tcm Cell Transcriptional Analysis
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Wild-type or Tcf7ΔGzmb CD62L+CD8+ Tcm cells were sort-purified from the primary recipients at 30–35 dpi and seeded onto flat bottom 96-well plate at 1×105 cells/well and incubated with GP33 peptide (200 nM) for 24 hrs, with dead cells removed using a Dead Cell Removal Kit (Miltenyi Biotec). The cells were used as GP33-stimulated CD8+ Tcm cells for RNA-seq and ATAC-seq analyses. For detection of IFN-γ production, Brefeldin A was added during the last 5 hrs of incubation. For mapping pathways leading to Tcf1 downregulation, Wild-type CD8+ Tcm cells were enriched with EasySep PE Positive Selection Kit (StemCell Technologies) after staining with CD62L-PE, and the cells were pre-incubated with pharmacological inhibitors or DMSO carrier for 30 min followed by 24-hr coculture with GP33. Benzoxathiole (Abcam, Cat. No. ab145954), Cyclosporin A, LY294002, and Selumetinib (Millipore-Sigma, Cat. No. 30024, 440202, and ADV465749271, respectively) were used at the final concentrations of 10 μM, 140 nM, 14 μM and 70 nM, which were equivalent to 10× their respective reported IC50 (Ref.29 (link), 30 (link)).
9
Tcf7ΔGzmb CD8+ Tcm Cell Transcriptional Analysis
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