FIG. 3.

Independent and lineage-specific expression of c-myb transcripts containing alternate exons during in vitro differentiation of CD34⁺ hematopoietic cells. (A) Differentiation protocol for CD34⁺ cells. Populations of primary human CD34⁺ cells were expanded (days 1 to 4) and then differentiated toward the myeloid or erythroid lineage (days 5 to 15) by using the indicated cytokines. SCF, stem cell factor; G-CSF, granulocyte colony-stimulating factor; Epo, erythropoietin. (B) Results from exon-specific qPCR analysis of differentiating CD34⁺ cells. RNA was isolated at the indicated time points, and qPCR was used to measure the levels of c-myb transcripts containing the indicated alternate exons. The graphs show the change (n-fold) in mRNA levels for the alternatively spliced c-myb variants, normalized by comparison to β2-microglobulin gene expression levels, relative to the mRNA levels in unstimulated CD34⁺ (day 0) cells. The solid vertical line on day 4 represents the transition of the cells from proliferation toward differentiation. The solid black lines indicate splice variant levels in cells undergoing myeloid differentiation, while the dashed black lines represent those in cells undergoing erythroid differentiation. For comparison purposes, the relative change (n-fold) in total c-myb mRNA levels was plotted on each graph and is represented by the gray lines. Exon 13A was not detectable after 10 days in culture. Error bars represent standard deviations of triplicate qPCR measurements. Similar results were obtained in two independent experiments. Note that the scale on the y axis is different in each graph to show the large variation in relative mRNA levels for the different c-myb splice variants. Alt exon, alternative exon.