FIG. 1.

Human C/EBPα mRNA and protein increase with granulocytic and shut off with monocytic differentiation. (a) Human C/EBPα mRNA is expressed specifically in cells of the myeloid lineage, as determined by Northern blotting of total RNA isolated from nonhematopoietic HeLa cells (lane 1), Jurkat T cells (lane 2), BJA-B (lane 3) and Raji (lane 4) B cells, promyelocytic Mono Mac 6 (lane 5), U937 (lane 6), and HL-60 (lane 7) cells, and K562 erythroleukemia cells (lane 8). The same blot was hybridized sequentially with a probe for human C/EBPα (hC/EBPα) and for 18S rRNA, as indicated to the right. Positions of migration of 28S and 18S rRNAs are marked on the left. Exposure was for 5 days with an intensifying screen. (b) The C/EBPα gene is activated at the stage of hematopoietic stem cell commitment to the myeloid lineage. Single human bone marrow cells were isolated as described in Materials and Methods and analyzed by Southern blotting of PCR-amplified cDNA. Shown are hybridization of the blot to a human C/EBPα-specific probe (top) and ethidium bromide staining of the gel before blotting (bottom). Each panel (bracketed on top) contains cDNA from five individual cells representing various stages of differentiation (marked above each bracket). Exposure was for 42 h with an intensifying screen. (c) C/EBPα is preferentially expressed in primary granulocytic cells, as determined by Northern blotting of total RNA purified from normal human peripheral blood monocytes and neutrophils. HeLa and HL-60 RNAs were included as negative and positive controls, respectively. The top panel shows results from hybridization to the human C/EBPα probe. Exposure was for 3 days with an intensifying screen. The same blot was stripped and rehybridized to an IL-8 receptor B (IL8RB; neutrophil-specific) probe (middle panel; 2-day exposure) and then to a 28S oligonucleotide as an internal control (bottom panel). (d) Expression of C/EBPα mRNA increases during granulocytic induction of myeloid cell lines and shuts off with monocytic differentiation. K562, U937, HL-60, and NB4 cells were stimulated to erythroid differentiation with DMSO, to monocytic differentiation with TPA, or to granulocytic differentiation with retinoic acid (RA); cell aliquots were withdrawn at the time points indicated above the blot, and RNA was analyzed by Northern blotting. Top, expression of human C/EBPα; middle, expression of the myeloid maturation marker CD18; bottom, 18S RNA as a control for RNA loading and quantitation of C/EBPα expression. Exposure was for 3 days (left) and 4 days (right) for C/EBPα and 4 days (left) and 18 h (right) for CD18, using an intensifying screen. (e) C/EBPα protein increases with retinoic acid-induced granulocytic induction of myeloid cell lines and decreases with TPA-induced monocytic differentiation. Shown is a Western blot of whole-cell extracts from uninduced U937 (lanes 1 and 11) and HL-60 (lanes 6 and 14) cells and retinoic acid (lanes 2 to 5 and 7 to 10)- or TPA (lanes 12, 13, 15, and 16)-induced cells. The cell lines and the inducers, as well as the times of induction in days, are indicated above the lanes. In the top panel, the blot was stained with anti-C/EBPα antibody; in the bottom panel, the same blot was stripped and subsequently stained with anti-β-tubulin antibody. Molecular size standards are shown in kilodaltons to the left. Arrows on the right indicate full-length C/EBPα and β-tubulin, and the asterisk indicates truncated C/EBPα protein (45, 53).