Effect of DPI on G1 phase cyclins and MK ploidy. (A) DPI decreases levels of G1 phase cyclins E and D3 in MKs. Wt BM was cultured (+TPO) for 3 days with or without 0.2 μM DPI. MKs were then purified from the cultures as in Figure 2, and lysates were subjected to Western blotting with the indicated antibodies. Data are representatives of 3 assays. An experiment performed with apocynin showed similar results (data not shown). (B) Expression of a cyclin E transgene partially restores the ploidy profile in Nox-inhibited MKs. Wt and cyclin E transgenic MKs were cultured (+TPO) for 3 days with 0.2 μM DPI or 200 μM apocynin. MKs were then subjected to ploidy analysis as described in “Methods” (data shown are representative of 3 experiments). (C) Cyclin E transgenic MKs have higher cyclin E levels compared with Wt on Nox inhibition. Wt and cyclin E transgenic MKs were cultured in the presence of 0.2 μM DPI and subjected to Western blot analysis with the indicated antibodies. Note that the levels of murine and human cyclin E are not comparable in this assay because they were probed with different antibodies. Similar increase in cyclin E levels in transgenic MKs compared with control was also observed in nontreated cells (not shown). Wt indicates wild-type MKs; cyclin E, cyclin E transgenic MKs. Data are representative of 3 assays.
