BCT-100 depletes arginine intracellularly. (A) Cell lines or patient samples were cultured with BCT-100 (600 ng/mL) for 72 hours. Intracellular arginine concentrations were measure by ELISA. BCT-100 causes a depletion of intracellular arginine. Data are representative of 2 independent experiments. (B) Internalization of BCT-100-AF647. AML blasts (top panel) and HL60 cells (bottom panel) were incubated with fluorescently labeled BCT-100 for 8 hours. Unbound drug was removed with stripping buffer and extensive washing. Nucleus was stained with 4,6 diamidino-2-phenylindole (DAPI). Images of representative cells were collected by LSM510 system (Zeiss). Arrows indicate intracellular localization of labeled BCT-100; arrowheads indicate surface-bound drug. Scale, 10 μm. Representative patient sample of 3 different patient samples.