Cytotoxicity assays are performed using the Alamar blue vital dye. For each cell line, preliminary dose?response curves are performed to establish the cell-concentration range, giving a linear relationship with fluorescence. For cell lines, 5,000 to 20,000 cells are plated in 200 μL complete medium in 96-well plates, in the presence or absence of increasing doses of drugs (0.01?10 μM). For ALL-2 and AML-PS leukemias 10 × 10⁵ cells/well are plated in StemSpanSFEM medium and treated with the same range of drug concentrations. Peripheral blood mononuclear cells and cord blood CD34+ cells are plated 1 × 10⁵ cells/well in presence or absence of 1 μg/mL phytohemagglutin or growth factor cocktail (50 ng/mL stem cell factor, 20 ng/mL each of granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interleukin-3, interleukin-6, and 3 U/mL erythropoietin), respectively. In all cases, after 48 hours culture, 1/10 volume Alamar blue solution is added and incubated overnight. The plates are then read in a fluorimeter with excitation at 535 nm and emission at 590 nm. Cytotoxicity is calculated as percentage of fluorescence with respect to untreated control, after subtracting for background fluorescence in absence of cells.

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

10⁷ HL60 and K562 cells are inoculated subcutaneously in SCID mice. Animals are randomized in seven mice per group. PHA-793887 is administered at 20 mg/kg intravenous (IV) once a day, continuously for 10 days (from day 9 to day 18) in HL60 model and with a two 5-day cycles (from day 9 to day 13 and from day 17 to day 21) in K562-bearing mice. Glivec is orally administered for 9 consecutive days from day 9 onward in the K562 xenograft model. Tumor growth and net body weight are evaluated twice a week. The tumor weight is calculated according to the following formula: tumor weight = length (mm) × width² (mm) /2. The effect of the anticancer treatment is determined as the delay in onset of an exponential growth of tumors. This delay (T ? C value) is defined as the difference of median time (in days) required for the tumors of treatment (T) and control groups (C) to reach a predetermined size. Toxicity is evaluated on the basis of the body weight reduction.

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

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