Fig. 1: Characterization of the Jak2-R1063H mouse model.

A Increased mortality rate of the mutant mice. Kaplan-Meier survival analysis of m/m (wt) (n = 15) and RH/RH (n = 15) mice. Mantle-Cox test was used to assess statistical significance of obtained results (p = 0.0350). B Increased baseline (blank line) and cytokine-stimulated Jak2/Stat5 activation in lysates of total BM cells obtained from RH/RH 3-month-old animals (n = 2, pooled cells), when compared to age-matched controls (n = 2), identified by WB analysis. C PLT counts for m/m (wt) and RH/RH are shown at the indicated times (up); comparison of PLT counts for m/m (wt), heterozygous and homozygous Jak2-R1063H mice at 3 and 12 months of age (down), n ≥ 10 mice per group, per each time point. D Phenotypic MkPs stratification based on CD48 expression. Representative flow cytometry plot (up) and quantification of CD48_low non-hierarchical MkPs at indicated time-points (n ≥ 3 mice per group) (down). E Tpo concentration in plasma (n ≥ 4 mice per group). F Workflow of transplantation of unsorted Jak2-R1063H-positive or wt BM cells to lethally irradiated C57BL/6NCrl recipients and PLT counts after transplantation (n ≥ 10 mice per group). G RBC, hemoglobin (HGB) and hematocrit (HCT) levels at the indicated times (n ≥ 10 mice per group, per each time point) (up). Comparison of RBC, HGB and HCT counts for m/m (wt), heterozygous and homozygous Jak2-R1063H mice at 3 and 12 months of age, n ≥ 10 mice per group, per each time point (down). H Epo concentration in plasma (n ≥ 4 mice per group). I Altered coupling of mouse (m) Jak2-R1063H or human (h) JAK2-R1063H to hematopoietic receptors. Mouse Jak2-Flag and human JAK2-Flag mutants were transiently expressed in HEK293 cells in which mouse V5-tagged Epor and human HA-tagged EPOR/TPOR were stably expressed. Interaction was examined by co-immunoprecipitation with Flag/V5 antibody. Immunoblot band intensity was quantified by ImageJ software and normalized to loading control and human JAK2 wt. All data are presented as mean ± SD and unpaired t-test with Welsch’s correction was used for group comparison. *p < 0.05.