Reaction mixtures contained 8 mM Tris-HCl (pH 7.5), 10 mM HEPES, 5 mM dithiothreitol, 10 μM ATP, 0.5 μCi ³³P-ATP, 10 mM MnCl₂, 150 mM NaCl, 0.01% Triton X-100, 4% DMSO, 0.05 mg/mL poly(Glu:Tyr) (4:1, average molecular weight of 20-50 kDa), and 7.5, 7.5, and 16 ng of FGFR1, FGFR3, and FGFR4, respectively, and are incubated at room temperature for 30 minutes followed by termination with 10% H₃PO₄. The reaction mixtures are transferred to 96-well MAFB filter plates that are washed 3 times with 0.5% H₃PO₄. After air-drying, the plates are read with a Trilux reader^[1].

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

The different multiple myeloma cancer cell lines KMS-11 and OPM-2 cells, L-363, and U266 cells are used. Cells (2,000 per well) are first grown in RPMI for 6 hours and treated with LY2874455 at 37°C for 3 days. The cells are stained at 37°C for 4 hours and then solubilized at 37°C for 1 hour. Finally, the plate is read at 570 nm using a plate reader^[1].

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

Mice^[1]
RT-112, OPM-2 (DSMZ), SNU-16, and NCI-H460 cells (RT-112: 2×10⁶ per animal; OPM-2: 10⁷ per animal; SNU-16: 10⁶ per animal; and NCI-H460: 3×10⁶ per animal) are mixed with Matrigel (1:1) and implanted subcutaneously into the rear flank of the mice (female, CD-1 nu/nu for RT-112, OPM-2, and NCI-H460 cells and female, severe combined immunodeficient for SNU-16 cells). The implanted tumor cells grow as solid tumors. To test the efficacy of LY2874455 in these models, the animals are orally dosed with approximately 1 mg/kg (TED₅₀) or 3 mg/kg (TED₉₀) of LY2874455 in 10% Acacia once (every day) or twice a day after tumors reach approximately 150 mm³. The tumor volume and body weight are measured twice a week.
Rats^[1]
Four male rats per group are dosed with vehicle (1% hydroxyethylcellulose, 0.25% polysorbate 80, and 0.05% Dow Corning antifoam 1510-US in purified water) on day 1 and LY2874455 (1, 3, and 10 mg/kg) on day 0. On day 1, at least 120 minutes of control data are collected following vehicle administration. On day 0, data are collected for approximately 20 hours beginning after the last animal is dosed.

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

• [1]. Zhao G, et al. A Novel, Selective Inhibitor of Fibroblast Growth Factor Receptors That Shows a Potent Broad Spectrum of Antitumor Activity in Several Tumor Xenograft Models. Molecular Cancer Therapeutics (2011), 10(11), 2200-2210.  [Content Brief]