Cystine dependency in AML. (A) AML cell lines, primary AML blasts (n = 4), and healthy cord blood (CB)-derived CD34⁺ stem/progenitor cells were grown in liquid culture conditions, and the effects on cell viability upon dose-dependent depletion of cystine were determined by flow cytometry with annexin V and propidium iodide (PI). (B) As in panel A, but effects of cell proliferation as determined by viable cell counts is shown. (C) The 50% effective concentration (EC50) values (mM) of experiments shown in panels A and B showing the effective concentration of cysteine at which a 50% reduction in viability or proliferation was achieved. Primary AMLs were measured on day 5, and all other samples on day 3 (n = 2-5 independent experiments performed in triplicate). Cell lines with the highest sensitivity to cystine depletion are marked in blue, and cell lines with the lowest sensitivity are marked in red. (D) Cell counts of CD34⁺/CD38⁻, CD34⁺/CD38⁺, CD34⁻/CD38⁺, and CD34⁻/CD38⁻ compartments upon cystine depletion. (E) Western blot for the levels of apoptosis markers cleaved poly ADP ribose polymerase and cleaved caspase 3 in HL60 and MOLM13 cells grown in the presence or absence of cystine for 20 hours; β-actin used as loading control. (F) Percent of viable (annexin V negative/PI negative), early apoptotic (annexin V positive/PI negative), late apoptotic (annexin V positive/PI positive), and nonviable (annexin V negative/PI positive) HL60, MOLM13, and K562 cells after 10 hours or 24 hours of growth in the absence of cystine.