Dose-dependent iron-chelating effects of ELT on megakaryopoiesis. (A) The iron-chelating effects of ELT were evaluated using the calcein iron assay in K562 cells. Free calcein fluorescence increased as intracellular free iron decreased in response to ELT in a dose-dependent manner. (B) ΔFluorescence, calculated as the maximal calcein fluorescence minus the fluorescence with a specific treatment, was used to reflect the intracellular LIP, which decreased with increasing ELT concentrations. Thirty µM ELT always induced the highest calcein fluorescence, which was used as maximal fluorescence for these calculations. Shown are the results for CB-MKs treated with ELT (n = 4), K562 cells treated with ELT (n = 3), and K562 cells treated with the intracellular iron chelator DFP (n = 3). (C-E) CB-CD34⁺ cells were cultured with TPO3 alone, TPO3 plus the indicated concentrations (µM) of ELT, or TPO3 plus the iron chelators DFO or DFP for 14 days. Cells were counted biweekly at the time of media changes. Compared with TPO3, ELT6 significantly stimulated cell growth (C), whereas ELT30 suppressed cell growth (D), similarly to DFO100 or DFP100 (E). Data shown represent the mean ± standard deviation (SD) of 4 independent cultures. **P < .01; ***P < .001.