Figure 4 - Depletion of mDia2 inhibits enucleation.

(a) Depletion of mDia1 and mDia2 in primary erythroid cells by shRNA. TER119-negative mouse fetal erythroblasts were infected with retrovirus vectors encoding an shRNA specific for mDia1 or mDia2, and cultured for 48 h. Lysates were subjected to western blotting for mDia1 or mDia2. Controls show GAPDH levels. (b) Flow cytometric analysis of cells infected with retroviruses encoding mDia1 or mDia2 shRNAs and cultured for 2 days, as in Fig. 1. Cells were stained with TER119–PE, FITC–CD71 and Hoechst 33342 and analysed by FACS. The percentages of R8 cells (reticulocytes) and TER119-negative and -positive cells are indicated. (c, d) Quantification of incipient reticulocytes in cells infected with indicated retrovirus (c) and the number of these cells (d). As in Fig. 2d, the total cell number is shown as grey bars and the normalized number as black bars. The error bars represent mean s.d. (n = 3). (e) Benzidine–Giemsa staining of 2 day-cultured erythroblasts infected with retrovirus expressing mDia2 shRNA. The arrowheads indicate cells blocked in the process of enucleation and the arrow indicates a binucleated cell. (f) Alexa Fluor 488–phalloidin staining of 2 day-cultured erythroblasts infected with retrovirus expressing mDia1 or mDia2 shRNA. The arrowheads indicate CAR in late-stage erythroblasts. The arrow indicates an incipient reticulocyte. The scale bar represents 6 m. (g) Quantification of cells containing a CAR from f. The error bars represent mean s.d. (n = 3).