Effects of IFN-α in Jak2V617F;Stat1−/− mice. (A) The number of CFU-GM from WT, Stat1−/−, Jak2V617F, and Jak2V617F;Stat1−/− mice in the presence of ropeg-IFN-α (0.01, 0.1, or 0.5 μg/mL). BM cells (2 × 104 per well) from each genotype were plated in methylcellulose containing erythropoietin, IL-3, stem cell factor, and IL-6. The numbers of CFU-GM were quantified after 7 days of culture. Each experiment was performed in duplicate, and 3 independent experiments were performed. Data are presented as mean ± SE of 3 experiments. P values were calculated by the Tukey test after 1-way ANOVA; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. (B) PB complete cell count after 8 weeks of ropeg-IFN-α treatment in Jak2V617F and Jak2V617F;Stat1−/− mice. Jak2V617F mice (n = 5 per PBS and n = 5 per ropeg-IFN-α treatment); and Jak2V617F;Stat1−/− mice (n = 10 per PBS and n = 11 per ropeg-IFN-α treatment); shown in panels B, D-E, respectively. Data are presented as mean ± SD. P values were calculated by the Tukey test after 1-way ANOVA; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. (C) Histological changes in the BM from Jak2V617F;Stat1−/− mice after 8 weeks of ropeg-IFN-α treatment. Top: HE staining (100×); middle: HE staining (400×); bottom: Ag staining (200×). A yellow arrowhead indicates a megakaryocyte. Mild BM fibrosis in Jak2V617F;Stat1−/− mice disappeared after 8 weeks of ropeg-IFN-α treatment. Lower, left: the grade of fibrosis according to 4 grades (n = 5 per each treatment group). Statistical significance was determined by Mann-Whitney U test for 2 groups. ∗P < .05. Lower, right: the average number of megakaryocytes in 5 HPFs (n = 5 per each treatment group). Data are presented as mean ± SD. P values were calculated by the Tukey test after 1-way ANOVA. (D) The decrement in the proportion of LT-HSCs and short-term HSCs (ST-HSCs) in the BM from Jak2V617F and Jak2V617F;Stat1−/− mice after 8 weeks of ropeg-IFN-α treatment. Data are presented as mean ± SD. P values were calculated by the Tukey test after 1-way ANOVA; ∗P < .05. (E) Analysis of the number of Jak2V617F and Jak2V617F;Stat1−/− HSCs and their subpopulations after 8 weeks of treatment with ropeg-IFN-α. Left: the total number of LT-HSCs in 2 femurs and 1 tibia from Jak2V617F and Jak2V617F;Stat1−/− mice. Right: percentage of CD41hi and CD41lo LT-HSCs. Orange bar, CD41lo LT-HSCs; dark orange bar, CD41hi LT-HSCs. Data are presented as mean ± SD. P values were calculated by the Tukey test after 1-way ANOVA. ∗P < .05. (F) PCA after 8-week treatment with ropeg-IFN-α, performed on Jak2V617F, Jak2V617F;Tyk2−/−, and Jak2V617F;Stat1−/− mice. PCA was integrated with 35 parameters described in the supplemental Methods. First from the left: PCA after PBS treatment on Jak2V617F, Jak2V617F;Tyk2−/−, and Jak2V617F;Stat1−/− mice; (second from the left) PCA with either PBS or ropeg-IFN-α treatment on Jak2V617F mice; (third from the left) PCA with either PBS or ropeg-IFN-α treatment on Jak2V617F;Tyk2−/− mice; (fourth from the left) PCA with either PBS or ropeg-IFN-α treatment performed on Jak2V617F;Stat1−/− mice. WBC, white blood cell; Hb, hemoglobin; Plt, platelet; n.s., not significant.

Effects of IFN-α in Jak2V617F;Stat1−/− mice. (A) The number of CFU-GM from WT, Stat1−/−, Jak2V617F, and Jak2V617F;Stat1−/− mice in the presence of ropeg-IFN-α (0.01, 0.1, or 0.5 μg/mL). BM cells (2 × 104 per well) from each genotype were plated in methylcellulose containing erythropoietin, IL-3, stem cell factor, and IL-6. The numbers of CFU-GM were quantified after 7 days of culture. Each experiment was performed in duplicate, and 3 independent experiments were performed. Data are presented as mean ± SE of 3 experiments. P values were calculated by the Tukey test after 1-way ANOVA; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. (B) PB complete cell count after 8 weeks of ropeg-IFN-α treatment in Jak2V617F and Jak2V617F;Stat1−/− mice. Jak2V617F mice (n = 5 per PBS and n = 5 per ropeg-IFN-α treatment); and Jak2V617F;Stat1−/− mice (n = 10 per PBS and n = 11 per ropeg-IFN-α treatment); shown in panels B, D-E, respectively. Data are presented as mean ± SD. P values were calculated by the Tukey test after 1-way ANOVA; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. (C) Histological changes in the BM from Jak2V617F;Stat1−/− mice after 8 weeks of ropeg-IFN-α treatment. Top: HE staining (100×); middle: HE staining (400×); bottom: Ag staining (200×). A yellow arrowhead indicates a megakaryocyte. Mild BM fibrosis in Jak2V617F;Stat1−/− mice disappeared after 8 weeks of ropeg-IFN-α treatment. Lower, left: the grade of fibrosis according to 4 grades (n = 5 per each treatment group). Statistical significance was determined by Mann-Whitney U test for 2 groups. ∗P < .05. Lower, right: the average number of megakaryocytes in 5 HPFs (n = 5 per each treatment group). Data are presented as mean ± SD. P values were calculated by the Tukey test after 1-way ANOVA. (D) The decrement in the proportion of LT-HSCs and short-term HSCs (ST-HSCs) in the BM from Jak2V617F and Jak2V617F;Stat1−/− mice after 8 weeks of ropeg-IFN-α treatment. Data are presented as mean ± SD. P values were calculated by the Tukey test after 1-way ANOVA; ∗P < .05. (E) Analysis of the number of Jak2V617F and Jak2V617F;Stat1−/− HSCs and their subpopulations after 8 weeks of treatment with ropeg-IFN-α. Left: the total number of LT-HSCs in 2 femurs and 1 tibia from Jak2V617F and Jak2V617F;Stat1−/− mice. Right: percentage of CD41hi and CD41lo LT-HSCs. Orange bar, CD41lo LT-HSCs; dark orange bar, CD41hi LT-HSCs. Data are presented as mean ± SD. P values were calculated by the Tukey test after 1-way ANOVA. ∗P < .05. (F) PCA after 8-week treatment with ropeg-IFN-α, performed on Jak2V617F, Jak2V617F;Tyk2−/−, and Jak2V617F;Stat1−/− mice. PCA was integrated with 35 parameters described in the supplemental Methods. First from the left: PCA after PBS treatment on Jak2V617F, Jak2V617F;Tyk2−/−, and Jak2V617F;Stat1−/− mice; (second from the left) PCA with either PBS or ropeg-IFN-α treatment on Jak2V617F mice; (third from the left) PCA with either PBS or ropeg-IFN-α treatment on Jak2V617F;Tyk2−/− mice; (fourth from the left) PCA with either PBS or ropeg-IFN-α treatment performed on Jak2V617F;Stat1−/− mice. WBC, white blood cell; Hb, hemoglobin; Plt, platelet; n.s., not significant.

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