The activity of MEK1 is assessed by measuring the incorporation of [γ-³³P]phosphate from [γ-³³P]ATP onto ERK2. The assay is carried out in a 96-well polypropylene plate with an incubation mixture (100 μL) composed of 25 mM HEPES (pH 7.4), 10 mM MgCl₂, 5 mM β-glycerolphosphate, 100 μM sodium orthovanadate, 5 mM DTT, 5 nM MEK1, 1 μM ERK2, and 0 to 80 nM selumetinib (final concentration of 1% DMSO). The reactions are initiated by the addition of 10 μM ATP (with 0.5 μC k[γ-³³P]ATP/well) and incubated at room temperature for 45 min. An equal volume of 25% trichloracetic acid is added to stop the reaction and precipitate the proteins. Precipitated proteins are trapped onto glass fiber B filter plates, excess labeled ATP is washed off with 0.5% phosphoric acid, and radioactivity is counted in a liquid scintillation counter. ATP dependence is determined by varying the amount of ATP in the reaction mixture.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Primary HCC cells are plated at a density of 2.0×10⁴?per well in growth medium. After 48 h in growth medium, the cell monolayer is rinsed twice with MEM. Cells are treated with various concentrations of Selumetinib (AZD6244, 0, 0.5, 1.0, 2.0, 3.0, and 4.0 μM) for 24 or 48 h. Cell viability is determined by the MTT assay. Cell proliferation is assayed using a bromodeoxyuridine kit as described by the manufacturer. Experiments are repeated at least thrice, and the data are expressed as mean±SE.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

To investigate the effects of Selumetinib (AZD6244) on HCC xenografts, AZD6244 is suspended in water at an appropriate concentration. Mice bearing HCC xenografts are p.o. given, twice a day, with either 100 μL of water (n=12) or 50 mg (n=12) or 100 mg (n=12) of AZD6244 per kilogram of body weight for 21 days, starting from day 7 after tumor implantation. Growth of established tumor xenografts is monitored at least twice weekly by Vernier caliper measurement of the length (a) and width (b) of the tumor. Tumor volume is calculated as (a × b²)/2. Animals are sacrificed 3 h after the last dose of ADZ6244, and body and tumor weights are recorded, with the tumors harvested for analysis.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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