Loss of TET2 interferes with cell-cycle progression and gene expression in JAK2V617F-LSKs. (A) DNA damage, cell cycle, and apoptotic status of mutant LSKs assessed by flow cytometry. Bone marrow CD45.2-LSKs from second recipients at 16 weeks after transplantation in a competitive transplantation assay were gated, and the proportion of DNA damaged, cycling, and apoptotic LSKs was assessed by γH2AX, BrdU, and cleaved poly (ADP-ribose) polymerase (PARP) positivity, respectively. BrdU labeling was assessed after 7 days of in-vivo incorporation. There was no difference in the degree of γH2AX-positive cells among the 4 types of mice. In terms of cell-cycle status, JAK2V617F-LSKs showed greatly increased cell cycling relative to WT-LSKs, but this increase was attenuated by loss of TET2, and the percentage of actively cycling cells eventually declined to the same level as WT-LSKs. Finally, TET2KD cells showed decreased apoptosis. (B) Dendrogram constructed from unsupervised hierarchical clustering of data sets from 4 types of BM-LSKs using Pearson correlation. (C) GSEA demonstrating positive enrichment of the STAT5A target genes in both JAK2V617F-LSKs and double-mutant-LSKs. The normalized enrichment score (NES) from the overall gene expression profiles of LSKs and the false discovery rate q-value are indicated. (D) GSEA demonstrating negative enrichment of the HSC signature genes in JAK2V617F-LSKs and double-mutant-LSKs. Error bars indicate standard deviation of representative experiments performed in triplicate. *P < .05 vs WT; †P < .05 vs JAK2V617F.