Lipid raft clustering is necessary for enucleation and is regulated by Rac GTPases. (A) Staining of control and NSC23766-treated enucleating erythroblasts with CTB-AlexaFluor-594, which labels the lipid raft marker ganglioside GM1, revealed that Rac inhibition inhibits lipid raft clustering in the cleavage furrow between incipient reticulocyte and pyrenocyte (indicated by white arrowheads). Three representative images are shown in each panel from at least 40 cells with similar morphology in each category. Images were obtained with a 60× objective lens by Imagestreamx. (B) The bright detail intensity of lipid raft staining decreased significantly in enucleating WT erythroblasts incubated with the Rac GTPases inhibitor NSC23766 (50μM). Medians and means for bright detail intensity of CTB staining in control WT and NSC23766-inhibited enucleating erythroblasts are shown, and the difference of the 2 samples is statistically significant with P < .001. (C) Effect of pharmacologic inhibitors of actin filament assembly (cytochalasin-D), lipid raft organization (filipin), and Rac GTPase activity (NSC23766) on the enucleating efficiency of WT erythroblasts in a fast enucleation assay as percentile of the control sample. The absolute enucleation efficiency of the control sample in this experiment was 51.5% ± 3.4%. Data are mean ± SEM; n = 6. *P < .05 (vs control). ‡P < .005 (vs control).
