EZH2 inhibition alters cell-cycle regulation in mature MKs. (A) Schematic diagram representing microarray analysis performed at day 9 of culture in presence of 2 different shEZH2 and GSK343 inhibitor (i). Principal-component (PC) analysis of microarrays performed at day 9 of culture (ii). Circles indicate the different cell populations; CD41⁺CD42⁺ cells are grouped in the red circle, CD41⁺CD42⁻ cells are grouped in the green circle; and CD41⁻CD42⁻ cells are grouped in the blue circle. (B) Heatmap showing genes up- or downregulated during MK differentiation. CD41⁻CD42⁻, CD41⁻CD42⁺, and CD41⁺CD42⁺ cell populations are shown. Missing values are in gray. (C) Volcano plot showing differentially expressed genes in the CD41⁺CD42⁺ cell population in CTL vs treated cells. (D) GO terms (biological process) differentially expressed in control cells (untreated and treated with a shCTL vs GSK343 and shEZH2, respectively). Corrected P values were calculated using a modified Fisher’s exact test followed by Bonferroni’s multiple comparison test. (E) List of the most downregulated genes (fold change $≥$1.5) in presence of EZH2 inhibition (GSK343 or shRNA). Gray squares indicate genes that belong to DNA replication pathway or DNA metabolic process according to MSigDB (Broad Institute). (F) GSEA of the CD41⁺CD42⁺ cells in presence of GSK343 and 2 shEZH2. FDR, false discovery rate; NES, normalized enrichment score.