Genetic deletion of all Rac GTPases results in impaired distribution of F-actin in enucleating erythroblasts. (A) Rac1^Δ/Δ;Rac2^−/−;Rac3^−/− erythroblasts exhibited statistically significant inhibition of enucleation in long-term in vitro erythropoiesis culture compared with WT erythroblasts (n = 3, P < .05 between WT and Rac1^Δ/Δ;Rac2^−/−;Rac3^−/− erythroblasts). Incubation of Rac1^Δ/Δ;Rac2^−/−;Rac3^−/− erythroblasts with 50μM NSC23766 did not enhance inhibition of enucleation in a significant manner, indicating that the inhibitory effect of NSC23766 in enucleation is probably not caused by off-target effects. (B) F-actin in Rac1^Δ/Δ;Rac2^−/−;Rac3^−/− erythroblasts continues to surround the nucleus in the enucleating erythroblast and does not assemble into CAR formation to separate the nucleus from the reticulocyte, in a similar pattern with the erythroblasts incubated with NSC23766. Images were obtained with a 40× objective lens by Imagestream^x.