Figure 7
Figure 7. Altered expression of erythropoietic and iron metabolism regulators in JAK2-Ex12 transgenic mice and erythroid precursors from PV patients with JAK2-Ex12 mutation. (A) Expression of mRNA for Mcl-1, Bcl-XL, Osm, and Tfr1 were determined by RT-PCR in MxCre;Ex12 mice, MxCre;V617F mice, or WT controls (n = 3 per group). One-way ANOVA with subsequent Bonferroni post-test was used. *P < .05. Mice were euthanized 12 weeks after pIpC injection. (B) Cell surface expression of Tfr1 (CD71) protein determined by flow cytometry. Representative flow cytometry plots of 1 mouse for each of the 3 genotypes (left). The geometric means of fluorescent intensities for CD71 are shown for the 3 genotypes (red, Ex12; blue, V617F; and black, WT) (middle). The quantification of the Tfr1 (CD71) cell surface expression from groups of 3 mice per genotype (right). Data are from 2 independent experiments. The mean of the CD71 fluorescent intensities ± SEM are shown as fold changes of the value found in WT mice. E12, MxCre;Ex12 and VF, MxCre;V617F. (C) Serum iron levels. (D) Liver iron levels (n = 6 mice per genotype). (E) Expression of mRNA for hepcidin (Hamp) determined by RT-PCR (n = 3 per group). (F) Hepcidin protein concentration (ng/mL) in the serum measured by ELISA (n = 3 per group). (G) Expression of mRNA for Erfe were determined by RT-PCR (n = 3 per group). (H) Hepcidin protein concentration in the serum of PV patients with JAK2 exon 12 mutations (n = 6) and in MPN patients with the JAK2-V617F mutation (PV, n = 10; ET, n = 12; and PMF, n = 10 patients per group). (I) Analysis of mRNA expression in BFU-E colonies from patients with PV. Peripheral blood mononuclear cells from 3 patients with JAK2 exon 12 mutations and 2 patients with JAK2-V617F were grown in methylcellulose, and single BFU-E colonies were picked and genotyped for JAK2 exon 12 mutations (red) or JAK2-V617F (blue), respectively. The mRNA from each colony was individually analyzed for TFR1 and ERFE expression by RT-PCR and the mean ± SEM values for all JAK2 (WT), heterozygous (het), and homozygous (hom) JAK2 mutant colonies are shown as the fold changes of the values obtained in WT colonies from the JAK2-V617F positive patients. The patients with JAK2 exon 12 mutations were only heterozygous, and homozygous colonies were not available. One-way ANOVA with subsequent Bonferroni post-test was used. *P < .05; ***P < .001. n.a., not available.

Altered expression of erythropoietic and iron metabolism regulators in JAK2-Ex12 transgenic mice and erythroid precursors from PV patients with JAK2-Ex12 mutation. (A) Expression of mRNA for Mcl-1, Bcl-XL, Osm, and Tfr1 were determined by RT-PCR in MxCre;Ex12 mice, MxCre;V617F mice, or WT controls (n = 3 per group). One-way ANOVA with subsequent Bonferroni post-test was used. *P < .05. Mice were euthanized 12 weeks after pIpC injection. (B) Cell surface expression of Tfr1 (CD71) protein determined by flow cytometry. Representative flow cytometry plots of 1 mouse for each of the 3 genotypes (left). The geometric means of fluorescent intensities for CD71 are shown for the 3 genotypes (red, Ex12; blue, V617F; and black, WT) (middle). The quantification of the Tfr1 (CD71) cell surface expression from groups of 3 mice per genotype (right). Data are from 2 independent experiments. The mean of the CD71 fluorescent intensities ± SEM are shown as fold changes of the value found in WT mice. E12, MxCre;Ex12 and VF, MxCre;V617F. (C) Serum iron levels. (D) Liver iron levels (n = 6 mice per genotype). (E) Expression of mRNA for hepcidin (Hamp) determined by RT-PCR (n = 3 per group). (F) Hepcidin protein concentration (ng/mL) in the serum measured by ELISA (n = 3 per group). (G) Expression of mRNA for Erfe were determined by RT-PCR (n = 3 per group). (H) Hepcidin protein concentration in the serum of PV patients with JAK2 exon 12 mutations (n = 6) and in MPN patients with the JAK2-V617F mutation (PV, n = 10; ET, n = 12; and PMF, n = 10 patients per group). (I) Analysis of mRNA expression in BFU-E colonies from patients with PV. Peripheral blood mononuclear cells from 3 patients with JAK2 exon 12 mutations and 2 patients with JAK2-V617F were grown in methylcellulose, and single BFU-E colonies were picked and genotyped for JAK2 exon 12 mutations (red) or JAK2-V617F (blue), respectively. The mRNA from each colony was individually analyzed for TFR1 and ERFE expression by RT-PCR and the mean ± SEM values for all JAK2 (WT), heterozygous (het), and homozygous (hom) JAK2 mutant colonies are shown as the fold changes of the values obtained in WT colonies from the JAK2-V617F positive patients. The patients with JAK2 exon 12 mutations were only heterozygous, and homozygous colonies were not available. One-way ANOVA with subsequent Bonferroni post-test was used. *P < .05; ***P < .001. n.a., not available.

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