Fig. 1

Effects of Nef on erythroid and myeloid cell development in vitro. Expression of Nef protein in CD34 + cells either transduced with LVX-vector or LVX-nef, or infected by NL43 was confirmed by Western blotting with a Nef specific antibody. Beta-actin was used as a loading control. The results are shown in a. CD34 and Nef (zsgreen1) expression and localization in the CD34 + cells transduced with LVX-nef was examined with immunofluorence staining and the results are shown in b. LVX-vector or nef transduced HSPCs were inoculated into methylcellulose-based media for colony forming assay. After 14 days in culture, numbers of CFU-GM, BFU-E and CFU-GEMM colonies were scored. Data are represented as mean ± SEM in c. At least 7 independent experiments were performed. Colonies formed in the methylcellulose-based media was shown in d. These colonies were harvested and some of the harvested cells were stained for CD45/CD235a (erythroid, e and f) and CD45/CD33/CD14 (myeloid, G and H) markers for flow analysis. Representative flow images of erythroid and myeloid lineage cells were shown in e and g, and the cumulative flow data of these two lineage cells were shown in f and h. The parental cells in f and h are referred to live cells gated in FSC/SSC plot and CD45 + cells, respectively. The rest of the harvested cells were subjected to cyto-spinning and Giemsa staining, and representative images of these cells are shown in i. Small arrows in i refer to erythroid cells at different developing stages. Data are represented as mean ± SEM. At least 3 independent experiments were performed. **p < 0.01; *p < 0.05