Ezh2 deletion alters gene expression in Jak2V617F HSCs. (A) Heat map showing significantly upregulated and downregulated genes in Jak2^VF/+ Ezh2^−/− LT-HSCs (Lin⁻Sca-1⁺c-kit⁺CD34⁻CD135⁻) compared with Jak2^VF LT-HSCs. (B) Venn diagrams comparing up- or downregulated genes in Jak2^VF/+ Ezh2^−/− LT-HSCs compared with Jak2^VF/+ or control LT-HSCs. (C) A list of selected genes that are significantly upregulated in Jak2^VF/+ Ezh2^−/− LT-HSCs compared with Jak2^VF/+ or control LT-HSCs. (D) Gene set enrichment analyses of the microarray data from control, Jak2^VF/+, and Jak2^VF/+ Ezh2^−/− LT-HSCs. Enrichment plots of selected gene sets with normalized enrichment score (NES) and false discovery rate (FDR) are shown. (E) Relative expression of S100a8, S100a9, Ifi27l2a, Ifit1, Cxcl10, Mpl, Ccl5, Xcl1, Pbx3, and Id2 mRNA was determined in control, Jak2^VF/+, and Jak2^VF/+ Ezh2^−/− LT-HSCs by quantitative real-time PCR and normalized with 18S expression. (F) Representative histograms on cell surface expression of Mpl (thrombopoietin [TPO] receptor) in control, Ezh2^−/−, Jak2^VF/+, and Jak2^VF/+ Ezh2^−/− mice BM megakaryocytes (CD41⁺CD61⁺) are shown on the left, and the fold change in median fluorescence intensity from 3 independent experiments are shown on the right as mean ± SEM. (G) Validation of some EZH2 targets in JAK2V617F-positive HEL cells. HEL cells were transduced lentiviral Ezh2 shRNA or scramble shRNA (control), and the infected cells were selected using puromycin. Relative expression of EZH2, S100A8, S100A9, IFI27 (homolog of mouse Ifi27l2a), CXCL10, CCL5, and ID2 was assessed by quantitative real-time PCR and normalized by glyceraldehyde-3-phosphate dehydrogenase. Data from 3 independent experiments are shown in bar graphs as mean ± SEM (*P < .05).