Stimulation of Hb synthesis in murine erythroleukemia cells. (A) DFP-mediated iron delivery to murine erythroleukemia (MEL) cells synthesizing hemoglobin is schematically depicted. (B) MEL cells suspended in serum-free DMEM containing 1.25 mg/mL bovine serum albumin and 5 mM HMBA were supplemented with various iron complexes (step 1 in panel A) and cultured for 48 hours. Hemoglobin synthesis (step 2 in panel A) was assessed in terms of hemoglobin content in cell lysates as described in “Cell hemoglobin synthesis” and is expressed relative to the hemoglobin content in control cells with no additions (set as 100%). The additions included 10 μM Fe complexed to 30 μM NTA without (Fe-NTA) and with 15 μM human apotransferrin (Fe:NTA + aTf); 10 μM Fe complexed to 30 μM DFP without (DFP-Fe) and with 15 μM human apotransferrin (DFP-Fe + aTf); 30 μM DFP alone (DFP); 15 μM human apotransferrin alone (aTf). Generation of holotransferrin from DFP and apotransferrin (DFP-Fe + aTf Dial.): 10 μM Fe:30 μM DFP complex was preincubated with 15 μM human for 1 hour and dialyzed. Results shown are averages of 3 separate experiments plus or minus SD; the average Abs₆₀₄ of control cells was 0.39. (C) DFP-mediated mobilization of iron from J774 (Fe donor; step 1a in panel A) cells to the extracellular medium (step 1b in panel A), followed by entry of DFP-Fe complexes into MEL (Fe acceptor) cells (step 1c in panel A) and intracellular donation of iron for hemoglobin (Hb) synthesis (step 2 in panel A). MEL cells were cultured for 48 hours in DMEM containing 1.25 mg/mL bovine serum albumin and 5 mM HMBA without (Con) or with 10 μM Fe-NTA (NTA-Fe), or with various supernatants of J774 cell lysates that were supplemented with 5 mM HMBA, and lysates were assayed for hemoglobin content. To obtain J774 mouse macrophage supernatants, J774 cells were cultured overnight without (untreated J774) or with 100 μM FAC (Fe-loaded J774), washed with 100 μM DFO to remove all extracellular iron, and incubated for 2 hours at 37°C in serum-free DMEM containing 1.25 mg/mL bovine serum albumin, without (−DFP) or with 30 μM DFP (+ DFP). The cell supernatants were collected and centrifuged to remove detached cells, and HMBA (5 mM) was added to MEL cells. Shown are values of hemoglobin (Hb; from a representative experiment) obtained in MEL cells exposed for 48 hours to the various conditions.