Crosstalk of iron and isocitrate with Epo signaling: PKCα/β as common target. (A-B) Epo levels influence isocitrate rescue. FACS analysis of 5 day cultures with indicated doses of Epo plus 25 ng/mL SCF; 100% or 15% transferrin, and 20mM isocitrate (IC) were included as indicated. Graph summarizes results of 3 independent experiments. (C) Iron restriction and Epo deprivation both induce PKCα/β hyperactivation. Epo levels influence the capacity of isocitrate to reverse the hyperactivation. Whole cell lysates from 4 day erythroid cultures underwent immunoblotting. (D) Kinetics of PKCα/β hyperactivation associated with erythroid iron restriction. (E-F) Complete reversal of viability defects by pan-PKC inhibitor. Erythroid cultures with 100% or 15% transferrin were treated with 0.5μM BIM, with FACS analysis on day 5. Graph represents 3 independent experiments. (G) Partial reversal of differentiation defects with selective PKC inhibitor. Cultures as in panel E were treated with either 0.5μM BIM or with 0.5μM Gö6983. Graphs summarize 3 independent experiments. DN indicates GPA⁻ CD41⁻ double-negative cells. **P = .02 for DN percentage in BIM + 15% versus 15%. **P = .01 for GPA percentage in Gö6983 + 15% versus 15%.