Effect of IFNα treatment on LT-HSC cell cycle in chimeric transplant recipients and primary mice. (A) Representative flow cytometry plots showing cell cycle in lineage^lowKit^highSca1⁺CD150⁺ WT (CD45.1) or Jak2^VF (CD45.2) LT-HSCs from chimeric recipient mice. G0 (quiescent) cells are Ki-67^lowH33342²ⁿ, G1 cells are Ki-67⁺H33342²ⁿ, and SG2M cells are Ki67⁺H33342⁴ⁿ. (B) Reduction in quiescent HSCs after IFNα treatment in WT (gated on CD45.1 WT vehicle: 40.3 ± 6.6% vs IFNα: 22.4 ± 6.0%; P < .01; n = 4) and reduction in quiescent HSCs after IFNα treatment in Jak2^VF (gated on CD45.2 Jak2^VF, vehicle: 28.0 ± 2.3% vs IFNα: 5.4 ± 0.7%; P < .0001; n = 5). There were significantly fewer quiescent Jak2^VF compared with WT HSCs (1.4-fold; P = .02). More profound depletion of quiescent Jak2^VF HSC compared with WT HSCs (4.2 fold; P = .0003). (C) Increase in actively cycling (Ki67⁺H33342⁴ⁿ) HSCs after IFNα treatment in Jak2^VF cells (gated on CD45.1 WT vehicle: 19.6 ± 0.4% vs WT IFNα: 24.2 ± 6.5%; P = .21; gated on CD45.2 Jak2^VF vehicle: 24.1 ± 0.9% vs Jak2^VF IFNα: 35.9 ± 9.6%; P < .05; n = 4-5). There were significantly more Jak2^VF than WT HSCs in active cell cycle in vehicle-treated recipients (1.2-fold; P < .01) and also in IFNα-treated recipients (1.5-fold; P = .05). Each data point represents an individual mouse. Data shown are representative of at least 2 independent experiments using chimeric recipient mice. Similar results were found in each experiment. Results given are mean ± standard deviation. (D) GSEA performed on lineage^lowKit^highSca1⁺CD150⁺ WT (CD45.1) or Jak2^VF (CD45.2) cells from chimeric recipient mice revealed that genes regulating cell cycle were enriched in Jak2^VF HSCs compared with WT HSCs. (E) Genes regulating quiescence were relatively enriched in WT HSCs compared with Jak2^VF HSCs. (F) Reduction in quiescent (Ki67^lowH33342²ⁿ) HSCs in WT and Jak2^VF primary mice after IFNα treatment (WT vehicle: 31.0 ± 3.2% vs WT IFNα: 21.7 ± 4.9%; P = .16; Jak2^VF vehicle: 22.0 ± 3.6% vs Jak2^VF IFNα: 11.1 ± 0.9%; P = .05; n = 3-4/group). (G) Increase in actively cycling (Ki67⁺H33342⁴ⁿ) HSCs in WT and Jak2^VF primary mice after IFNα treatment (WT vehicle: 20.0 ± 1.1% vs WT IFNα: 31.7 ± 2.6%; P < .01; Jak2^VF vehicle: 30.1 ± 1.6% vs Jak2^VF IFNα: 41.3 ± 1.4%; P < .01; n = 3-4/group). There were significantly more Jak2^VF than WT HSCs in active cell cycle in vehicle-treated recipients (P < .01) and also in IFNα-treated recipients (P = .03).