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. 2003 Apr 15;17(8):959–964. doi: 10.1101/gad.251903

Figure 4.

Figure 4

Functional differences between SCL protein isoforms in hematopoietic lineage outcome. (A) HEL cells were infected with the empty pMSCVpuro vector (−), or vectors encoding the SCL-A or SCL-D isoform. After puromycin selection cells were analyzed for expression of the megakaryocytic surface marker CD41a by flow cytometry. Data shown are the mean ± S.D. of two independent infections with analysis performed in duplicates. All HEL cells expressed similar amounts of recombinant proteins (data not shown). (B) Mouse bone marrow cells were infected with the empty pMSCV vector (−), or vectors encoding SCL-A (p42) or SCL-D (p22) isoforms. Recombinant SCL was detected in nuclear extracts by immunoblotting with a monoclonal antibody against human SCL. The blot was stripped and reprobed with an anti-E12 antibody as a control. (C) Increase of megakaryocyte colonies by full-length SCL-A. Mouse bone marrow cells were infected with the indicated retroviral constructs, antibiotic (G418)-selected, and plated in methylcellulose culture under selective pressure. Colonies were scored on day 9 of culture. Three independent experiments performed in duplicates are shown for each: control (open circles), SCL-A (gray circles), and SCL-D (black circles); the mean is indicated by a bar. The differences between control, SCL-A, and SCL-D are significant (p < 0.05). (D) Increased numbers of erythroid progenitors in multipotent colonies by SCL-D. Clonal analysis of erythroid development from multipotent progenitors. Mouse bone marrow cells were infected with the indicated retroviral constructs, antibiotic (G418)-selected, and plated in methylcellulose culture supplemented with erythropoietin. On day 10, pluripotent colonies were individually picked and assayed for their contents in erythroid progenitors (CFU-Es). The pluripotent colonies were ranked according to their capacities to give rise to increasing numbers of CFU-Es as shown. Note that SCL-D colonies show the highest incident of CFU-Es. Data shown are typical of two independent experiments.