Cell Culture

K562 wild type cells (American Type Culture Collection, Manassas, VA, USA) were cultured in a RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS)(Sigma-Aldrich, St. Louis, MO, USA) at 37˚C, 5% CO₂. K562 wild type cells were incubated with IM (initial at 0.05 µM) to establish IM resistance using the step-wise method described previously ¹⁴. IM (Sigma, USA) concentration was increased by 0.05 µM every ten or twenty days until reaching 2.8 µM. The surviving cells were then cultured in RPMI-1640/10% FBS at 37˚C, 5% CO₂ in the absence of IM for approximately three days. Cells were re-examined for sensitivity to IM and resistant K562 cells were labeled as K562-R. K562-R-EphB4-sh represented the established IM resistant cells with EphB4 knockdown by shRNA from our previous study ¹⁴.