Implication of aconitase activity in iron regulation of erythropoiesis. (A) Erythroid-specific aconitase inactivation during iron restriction. Enzymography (Acon Activity) was performed on extracts from day 4 cultures in erythroid (Ery), megakaryocytic (Mk), or granulocytic (Gran) media with 100% or 15% transferrin saturation; controls consisted of K562 cells expressing shRNAs targeting ACO2 or ACO1. Expression of mitochondrial aconitase protein in samples (bottom panel, IB: ACO2). (B) Summary of 3 independent experiments as in panel A. The y-axis is the ratio of scanned signals obtained with 15% transferrin saturation to those obtained with 100% transferrin saturation, expressed as mean percentage ± SEM. **P < .001 for Ery ACO1 changes and 0.03 for Ery ACO2 changes. Mk and Gran signals showed no significant changes. (C) Direct aconitase inhibition impairs erythroid differentiation in a manner similar to iron restriction. Erythroid cultures with 100% transferrin saturation were supplemented with fluorocitrate (FC) and underwent FACS assessment of differentiation (top panels) and viability (forward [FSC] and side scatter [SSC] in bottom panels) on day 5. Where indicated, 10mM citrate was also included in culture. (D) Summary of 3 independent experiments as in panel C, using 200μM fluorocitrate.