American Society of Hematology

Fig. 1.

$Fig. 1. Presence of cell-cycle proteins in 2N/4N and at least 8N human megakaryocytes. / (A) Flow cytometric analysis of day-8 megakaryocytes derived from G-CSF–mobilized blood CD34+ after labeling with the Hoechst 33342 dye and a PE–anti-CD41 mAb. CD41+ cells were sorted into 2N/4N and into greater than 4N cell fractions. Hoechst staining was analyzed by means of a linear scale. (B) Western blot analysis of cyclin (Cyc) D3, cyclin E, cyclin A, p21, p27, and p16 from the 2 sorted cell populations. The same quantity of proteins was loaded in the 2N/4N and at least 8N lanes. (C) Comparison of the expression of cyclin D3 and cyclin B1 by Western blots from the 2 sorted cell populations. This experiment was perfomed with a pool of sorted cells different from that used to generate the analysis in panel B. The same quantity of protein was loaded in the 2N/4N and at least 8N lanes, as confirmed with the use of actin as a control. In these 2 pools of sorted cells, purity of the cells of at least 8N was greater than 95%, and composed of two thirds of 8N cells and one third 16N cells.$

Presence of cell-cycle proteins in 2N/4N and at least 8N human megakaryocytes.

(A) Flow cytometric analysis of day-8 megakaryocytes derived from G-CSF–mobilized blood CD34⁺ after labeling with the Hoechst 33342 dye and a PE–anti-CD41 mAb. CD41⁺ cells were sorted into 2N/4N and into greater than 4N cell fractions. Hoechst staining was analyzed by means of a linear scale. (B) Western blot analysis of cyclin (Cyc) D3, cyclin E, cyclin A, p21, p27, and p16 from the 2 sorted cell populations. The same quantity of proteins was loaded in the 2N/4N and at least 8N lanes. (C) Comparison of the expression of cyclin D3 and cyclin B1 by Western blots from the 2 sorted cell populations. This experiment was perfomed with a pool of sorted cells different from that used to generate the analysis in panel B. The same quantity of protein was loaded in the 2N/4N and at least 8N lanes, as confirmed with the use of actin as a control. In these 2 pools of sorted cells, purity of the cells of at least 8N was greater than 95%, and composed of two thirds of 8N cells and one third 16N cells.

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