Figure 2 - Deregulation of Rac GTPases blocks enucleation.

(a, b) Effect of overexpression of mutants of Rho, Rac and CDC42 on formation of reticulocytes during in vitro culture. TER119-negative mouse fetal erythroblasts were infected with bicistronic retroviruses encoding CD4 and different Flag-tagged mutant forms of several Rho-family GTPases, or as control encoding human CD4 alone. Cells were analysed by flow cytometric analysis after 48 h in culture and the percentages of incipient reticulocytes were quantified. Grey bars indicate the uninfected hCD4-negative cells that provide an internal control, and the black bars indicate hCD4-positive cells expressing different Rho GTPase mutants. (c) Cells from a and b were harvested after 48 h in culture and analysed by western blot using anti-Flag antibodies. (d) TER119-negative mouse fetal erythroblasts were cultured as in Fig. 1. After 35 h, the indicated amount of NSC23766 was added. Cells were harvested after 48 h and analysed by flow cytometry and Benzidine–Giemsa staining. The percentages of R8 cells (reticulocytes) are indicated. The scale bar represents 12 m. (e) Cells from d were harvested after 48 h in culture. Total levels of Rac1/2, RhoA and Cdc42 were determined by western blot using the indicated antibodies. The activities of Rac, RhoA and Cdc42 were determined by GTPase activity assay. (f) The same cells as in c were analysed by flow cytometry for their differentiation status. The percentages of TER119-negative and positive cells are indicated. (g) The same cells as in c were counted and the total number is shown (grey bars). The number was normalized (see main text) and is shown as black bars. (h) TER119-negative mouse fetal erythroblasts were cultured as in Fig. 1. After 35 h in culture, 100 M NSC23766 was added to some cells and after 48 hours in culture the erythroblasts were stained with Alexa Fluor 488–phalloidin. The scale bar represents 8 m. (i) Quantification of results from h. All error bars represent mean s.d. (n = 3).