Enhanced activation of HSCs upon poly(I:C) challenge in NFAT5-deficient mouse models. (A) Representative flow cytometry dot plots illustrating the gating strategy, ESAM expression, and cell cycle analysis for HSCs (starting from a population depleted of lineage⁺ cells with an antibody cocktail against TER119, CD3, B220, CD11b, and Ly6G) in mice left untreated or injected with poly(I:C). Treatment was two doses of 2.5 mg/kg poly(I:C) injected 48 h apart, followed by analysis of the mice 24 h after the second injection. (B) Percentage of LSK and MPP3/4 populations in the bone marrow of untreated or poly(I:C)-treated Nfat5^+/+ Vav-Cre and Nfat5^fl/fl Vav-Cre mice. (C) Percentage of quiescent HSCs in untreated or poly (I:C)-treated Nfat5^+/+ and Nfat5^fl/fl Vav-Cre mice. Results in B and C are from two independent experiments, comprising a total of three untreated and nine poly(I:C)-treated mice of each genotype. (D) RT-qPCR analysis of mRNA expression of different ISGs in HSCs sorted from poly(I:C)-treated (2.5 mg/kg, 24 h) Nfat5^+/+ and Nfat5^fl/fl LysM-Cre mice. Results are from three independent pairs of wild-type and NFAT5-deficient littermate mice. (E and F) Flow cytometry analysis of the percentage of LSK and MPP3/4 populations (E) and percentage of quiescent HSCs (F) in untreated or IFNα-treated (100,000 U/mouse, 24 h) Nfat5^+/+ Vav-Cre and Nfat5^fl/fl Vav-Cre mice. Results show three untreated mice of each genotype, and eight wild-type and seven NFAT5-deficient IFNα-treated mice. Each dot represents an independent mouse. Empty dots in B–F represent wild-type mice and solid dots are NFAT5-deficient mice. Statistical significance was determined with an unpaired t test. *, P < 0.05; **, P < 0.01.