Loss of Tet2 does not reverse JAK2V617-induced hyperproliferation in single HSCs. (A) Table summarizing the mutations in each genotype and the color code for the remainder of the figures. (B-C) The cumulative times to first (B) and second (C) division are shown and were determined by manual daily cell counts, scoring the presence of 1, 2, or 3 to 4 cells. Loss of Tet2 on its own (green line) does not affect cell cycling compared with WT (blue line), whereas JAK2V617F on its own (red line) increases proliferation (P < .0001). Compound mutants (JAK HOM TET HOM, orange line) behave like JAK2V617F single-mutant HSCs with a faster time to first and second division, respectively (P < .0001). Data are shown as mean ± SEM. WT, n = 572; JAK HOM, n = 439; TET HOM, n = 566; JAK HOM TET HOM, n = 435; and are from 3-6 independent experiments. (D) Colony size after 9 days in culture. JAK2V617F and double-mutant HSCs give rise to a higher frequency (P < .0001 and < .0001) of large colonies compared with WT controls. Colonies were categorized as very small (>50), small (51-500), medium (501-10 000), or large (>10 000). WT, n = 572; JAK HOM, n = 439; TET HOM, n = 566; JAK HOM TET HOM, n = 435. (E-F) After 10 days in culture, colonies were analyzed by flow cytometry for differentiation markers. Colonies with JAK2V617F (JAK HOM [red bars] and JAK HOM TET HOM [orange bars]) have an increased frequency of Lin⁺ cells (Ly6g⁺, Mac1⁺; JAK HOM, P < .0001; JAK HOM TET HOM, P = .0177). Double mutants (orange bars) and JAK HOM (red bars) show a reduced frequency of KSL (Ly6g⁻, Mac1⁻, c-Kit⁺, Sca1⁺) cells compared with WT (blue bars) and TET HOM (green bars; JAK HOM TET HOM, P = .001; JAK HOM, P < .0001). WT, n = 165; JAK HOM, n = 178; TET HOM, n = 154; JAK HOM TET HOM, n = 145 (3 independent experiments).