C/EBPα switches lineage potential of MEL and EML cells toward myeloid development. (A) MEL cells were transduced by retrovirus containing MSCV-C/EBPα-GFP or MSCV-GFP as described in “Materials and methods.” After sorting GFP⁺ cells, MEL cells were cultured in vitro for an additional 3 days, after which cell morphology was determined by Wright-Giemsa staining, and cell lineage by naphthol AS-D chloroacetate esterase and MPO staining (× 1000). Arrows indicate stained myeloid granules. (B) RT-PCR was performed using total RNA harvested from the cultured MEL cells 3 days after transduction with retrovirus containing MSCV-C/EBPα-GFP or MSCV-GFP. (C) The SCF-dependent EML cell line (gift of Dr Schickwann Tsai) was maintained in IMDM supplemented with 20% horse serum, 8% conditioned medium from BHK/MKL cells containing mSCF, and P/S/G. EML cells were cultured with mSCF (100 ng/mL) and hEpo (40 U/mL) 24 hours prior to transduction with retrovirus containing pMSCV-C/EBPα-GFP or pMSCV-GFP. EML cells were transduced with the retroviral vectors that express MSCV-C/EBPα-GFP or MSCV-GFP and then cultured for 3 days with mSCF (100 ng/mL) and hEpo (40 U/mL) to promote erythroid development. The expression of (GFP⁺) granulocytes and monocytes (Gr-1 and Mac-1) and erythroid cells (CD71 and TER119) was determined by flow cytometry.