IMiDs inhibit proplatelet formation by blocking estradiol action. (A) Gene expression profiles of untreated and lenalidomide (Len)-treated megakaryocytes to examine genes involved in the terminal differentiation of megakaryocytes and platelet production was evaluated by gene set enrichment analysis (GSEA). (B) GSEA comparing untreated and Len-treated megakaryocytes for estradiol-upregulated (left panel) and -downregulated (right panel) genes. For each gene set, an enrichment score was calculated using the normalized Kolmogorov-Smirnov statistic. To adjust for the different sizes of gene sets, normalized enrichment score (NES) was calculated. (C-D) Representative phase-contrast microscopic images of megakaryocytes cultured with or without 10 µM of Len (C) or pomalidomide (Pom) (D) alone or in the presence of 10 nM of estradiol. Arrows denote megakaryocytes with proplatelets. Insets show enlarged images. Images were captured by BZ-X700 (Keyence) with ×20 (large images; scale bars, 50 µm) and ×40 (inset images; scale bars, 25 µm) objective lens. Mean percentages of proplatelet-forming megakaryocytes in 500 cells analyzed per sample are shown in the right panels. Data are from 3 independent experiments and expressed as means ± standard errors of the mean. P values were determined by 2-tailed Student t test.