Effect of IFN-α on the gene expressions of stem/progenitor cells in Jak2V617F and Jak2V617F;Tyk2^−/− mice. (A) Gene set enrichment analysis (GSEA) of genes regulating cell cycle (negative regulation of cell cycle G1 S phase transition) and genes regulating antiproliferative responses of IFNs. Upper: negative enrichment of genes for negative regulation of cell cycle G1 S phase transition in Jak2V617F CD150⁺LSK cells after IFN-α treatment but not in Jak2V617F GMPs. The gene expression data in CD150⁺LSK cells and GMPs in each genotype mice were obtained from 4 weeks of PBS- or ropeg-IFN-α–treated Jak2V617F and Jak2V617F;Tyk2^−/− mice. Jak2V617F treated with PBS (n = 3), Jak2V617F treated with ropeg-IFN-α (n = 4), Jak2V617F;Tyk2^−/−treated with PBS (n = 4), and Jak2V617F;Tyk2^−/− treated with ropeg-IFN-α (n = 5). Lower: enrichment of genes regulating antiproliferative responses of IFNs in Jak2V617F GMPs but not in Jak2V617F CD150⁺LSK cells. Genes regulating the antiproliferative responses of IFNs are listed in supplemental Table 3. GSEA was performed using a false discovery rate (FDR) cutoff of ≤0.25. Enrichment plots for each gene set are shown with NESs and FDRs. (B) Representative flow cytometry plots showing the cell cycle in CD150⁺LSK cells and GMPs after 2 weeks of PBS or ropeg-IFN-α treatment in Jak2V617F and Jak2V617F;Tyk2^−/− mice. G0 (quiescent) cells were Ki-67^lowH33342,²ⁿ G1 cells were Ki-67⁺ H33342,²ⁿ and SG2M cells were Ki67⁺H33342⁴ n. (C) Effect of IFN-α treatment on cell cycle of CD150⁺LSK cells and GMPs in Jak2V617F and Jak2V617F;Tyk2^−/− mice. Upper: reduction in quiescent CD150⁺LSK cells (Ki-67^lowH33342²ⁿ) and increase in actively cycling CD150⁺LSK cells (Ki67^lowH33342²ⁿ) after IFN-α treatment in Jak2V617F mice. These IFN-α effects on CD150⁺LSK cells were absent in Jak2V617F;Tyk2^−/− mice. Lower: reduction in actively cycling GMPs (Ki67⁺H33342⁴ⁿ) after IFN-α treatment in Jak2V617F mice but not in Jak2V617F;Tyk2^−/− mice. BM cells from 3 mice of each type were mixed, and cell cycle analysis was performed. The sample size for each treatment group was 3. Results are shown as mean ± SD of 3 samples. (D) Normalized expression levels of selected genes that regulate the antiproliferative responses to IFN-α. CD150⁺LSK cells and GMPs were isolated from the BM of Jak2V617F and Jak2V617F;Tyk2^−/− mice treated with 600 ng of ropeg-IFN-α for 4 weeks. After normalization to the expression levels of Gapdh, the expression levels of each gene in each mouse genotype are shown as relative ratios to those in WT mice. Results are shown as mean ± SD of 3 samples. Statistical significance was determined using the Tukey test after 1-way ANOVA; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. NES, normalized enrichment scores; n.s., not significant.