FIGURE 1.

5-Aza-CdR stimulated the expression of genes associated with erythroid iron acquisition and heme synthesis. MEL cells were treated with 1.8% DMSO or 5-aza-CdR (0.1 or 1 μm) for 48 h. Cells were collected for total RNA and protein isolation. A, quantitative real-time RT-PCR showed increases in the mRNA levels of TfR1, Hba1, Hbb1, Alas2, and Fech. *, p < 0.01 compared with the control MEL cells. B, a representative Western blot showing the levels of TfR1, Alas2, and Fech protein in 5-aza-CdR-treated MEL cells. C, densitometry analysis of the expression of TfR1, Alas2, and Fech protein levels (the densitometry was normalized to the levels of β-actin). *, p < 0.01 compared with the control MEL cells. D, cellular iron uptake in control and 5-aza-CdR-treated MEL cells. MEL cells treated with or without 5-aza-CdR for 48 h were incubated for 0.5, 1.5, or 3 h with 1.85 μm ⁵⁵Fe-Tf. After washing three times with cold PBS, the radioactivity was measured using a scintillation counter. *, p < 0.01 compared with 1.5 h control MEL cells; **, p < 0.01 compared with 3 h control MEL cells. E, heme levels in MEL cells treated with or without 5-aza-CdR. Cellular extracts (10 μg of total protein) were analyzed by measurement of fluorescent PPIX levels after removing iron from endogenous heme. *, p < 0.01 compared with control MEL cells. Error bars, S.E.