Iron-limited erythroid differentiation of MEL cells. (A) Timeline depicting the experimental time course of DMSO-induced erythroid differentiation of MEL cells under normal conditions or iron-deficient conditions induced by the iron chelator DFO. (B) Electrophoretic mobility shift assay of MEL cell protein extracts using a 32P-labeled ferritin IRE probe showed activation of IRP1 and IRP2 after treatment with DFO. (C) Aconitase activity gel demonstrating a time-dependent increase in mitochondrial (m-) and cytosolic (c-) aconitase activity levels over the course of differentiation, which was attenuated in DFO-treated cultures. (D) ALAS2 mRNA was induced during differentiation in the presence or absence of DFO. However, ALAS2 protein expression (E) was repressed in DFO-treated cells. Sample loading was assessed by reprobing for actin mRNA and protein, respectively. (F) Mature (heme-containing) hemoglobin (Hb) formation was repressed in differentiating MEL cells treated with DFO. Hemoglobin was measured by a modified diaminobenzidine procedure after separation of total cellular protein (40 μg) by native PAGE followed by transfer to polyvinylidene difluoride filters (“Hemoglobin assay”).
