Studies of hCdh1 and cyclin B1 expression in megakaryocytes.

Immunofluorescence was performed on day 6 megakaryocyte culture as described in Figure 5. (A) HCdh1 immunodetection in human megakaryocytes. (i) Interphase polyploid megakaryocyte shows a nuclear hCdh1 staining, using a polyclonal rabbit antibody followed by donkey TRITC-labeled F(ab′)2 fragments. DNA was counterstained with DAPI. (ii) Double immunostaining of hCdh1 and tubulin with DNA painting. HCdh1 was localized at the spindle poles during endomitosis. Bar 10μm. (B) Timing of cyclin B1 degradation in endomitosis. Cyclin B1 was immunodetected by using a mouse antibody followed by incubation with TRITC-conjugated antimouse F(ab′)2 fragments. DNA was labeled with DAPI. Three sequential phases of endomitosis were observed on a single view: G2/M megakaryocyte in the middle with a strong nuclear staining, prometaphase megakaryocyte in the lower-left corner with cyclin B1 localized at the spindle poles, and anaphase in the upper-right corner in which cyclin B1 is undetectable. Bar, 10 μm.