Rescue of U0126-induced enucleation impairment by vacuolin-1. (A) Representative western blot showing changes in pERK1/2 and ERK1/2 expression under various conditions as indicated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. (B) Representative flow cytometry analyses of enucleation under various conditions as indicated. (C) Quantitative analyses of enucleation from 3 independent experiments. (D) Representative cytospin images of orthochromatic erythroblasts under various conditions as indicated. Scale bar, 10 μm. (E) Quantitative analyses of enucleation from cytospin images. A total of 100 cells of each group from 3 independent experiments were used for quantification. (F) Representative EM images of orthochromatic erythroblasts under different conditions as indicated. White arrows indicate vacuoles. Scale bar, 5 μm. (G) Quantification of erythroblasts harboring vacuoles with a size >1 μm². A total of 100 cells of each group from 3 independent experiments were used for the quantification. (H) Quantitative analysis of the average area of intracellular vacuoles in erythroblasts. A total of 100 cells of each group from 3 independent experiments were used for quantification. Data are expressed as mean ± standard deviation. *P < .05, **P < .01, ***P < .001. FSC, forward scatter.