C/EBPα inhibits erythroid differentiation of MEL cells. (A) MOCK and C/EBPα/hER-transduced MEL clones were incubated in the presence of HMBA (3 mM) and β-estradiol (1 μM) for 96 hours. Red cell pellets indicate terminal differentiation of MEL cells. C/EBPα/hER and α-actin protein expression of each clone was confirmed by Western blot analysis as described in “Materials and methods.” (B) Hemoglobinization of MEL, MOCK, and αER-E4 cells was evaluated at the indicated times after treatment with (i) HMBA or (ii) HMBA + β-estradiol by benzidine staining. The mean benzidine positivity of each cell group ± SE of triplicate experiments is shown. (C) β-Globin mRNA levels were measured in HMBA-treated MOCK and αER-E4 cells by Northern blot analysis as described in “Materials and methods” using 447-bp fragment of mouse β-globin cDNA. Control lanes show 18S and 28S ribosomal RNA.