Regulation of EpoR and Id1 expression by C/EBPα during erythroid differentiation. (A) MEL cells, MOCK cells, and αER-E4 cells were cultured in 3 mM HMBA and/or 1.0 μM β-estradiol as indicated. Id1 protein expression was determined 24 hours after treatment by Western blot analysis as described in “Materials and methods.” (B) MEL cells were transduced with control (pMSCV-GFP) or Id1-expressing (pMSCV-Id1-GFP) retroviral vectors. Stable cell lines were cloned and Id1 expression was determined by Western blot analysis. Asterisks indicate the cell lines used for induction of erythroid differentiation. (C) Cloned MEL cells were seeded at 4 × 10⁵/mL in cDMEM containing 3 mM hexamethylene bisacetamide (HMBA) and incubated at 37°C. Erythroid differentiation of Id1-expressing cell lines was determined by benzidine staining 4 days after HMBA treatment (3 mM). (D) Total RNA was obtained from MOCK and αER-E4 cell clones at the indicated times after treatment with HMBA (3 mM) and β-estradiol (1 μM). EpoR mRNA expression in MOCK and αER-E4 cells was analyzed by Northern blot analysis before and after treatment with HMBA and β-estradiol. The EpoR probe was an XhoI 1450-bp fragment of the murine EpoR cDNA (gift of Dr Sandy Ruscetti, NCI-Fredrick. (E) Total cellular extracts were prepared from C/EBPα^–/–, C/EBPα^+/–, C/EBPα^+/+ FL cells, and EpoR expression was determined by Western blot analysis of 30 μg protein. (F) L cells were transfected with the pGL3, 2xC/EBPα-Luc, mEpoR-Luc, alone or with increasing amounts (20, 50, 100 ng) of C/EBPα expression vector by liposomal method (FuGENE 6). Cell extracts were assayed after 48 hours of incubation with complete DMEM media. The histograms represent means ± SE of the luciferase activity from triplicates normalized for transfection efficiency using renilla luciferase. **P < .05 t test.