Isocitrate abrogation of the erythroid iron-restriction checkpoint. (A) Isocitrate (IC) reverses defects in differentiation and viability. Five-day erythroid cultures with 15% transferrin saturation included trisodium isocitrate (IC) at 5mM. FACS analysis of GPA and CD41 expression with gating on viable fraction. (B) Summary of 3 independent experiments as in panel A. (C) Isocitrate reverses structural changes associated with iron restriction. Light microscopy of Wright-stained cytospins from cultures in panel A (400× magnification). Images were acquired with the use of an Olympus BX51 microscope equipped with an Olympus DP70 digital camera. The objective lens consisted of Uplan Fl 40×/0.75 NA. Image acquisition and processing used Adobe Photoshop, CS3/10.0 and CS2/9.0, respectively. (D) Isocitrate enhances hemoglobinization. Erythroid cultures with 100% or 15% transferrin saturation were supplemented with 20mM isocitrate. Photograph of day 5 cell pellets. (E) Isocitrate augments globin chain expression. Whole cell lysates from panel D underwent immunoblotting. (F) IRE-binding activity of IRP1 and IRP2: influences of transferrin saturation and isocitrate. RNA gel-shift assays were performed on extracts from day 4 CD34⁺ cultures in erythroid medium with 100% or 15% transferrin saturation and 20mM isocitrate. (G) IRP2 stabilization by iron deprivation: minimal effect of isocitrate. Cellular extracts from panel F underwent immunoblotting. FSC indicates forward scatter; and SSC, side scatter.