2.8. Statistics

Data are presented as mean ± s.d. Statistical significance was assessed using Student’s t-test for comparisons between two groups and ANOVA for differences between groups. The p-values wherever applicable (Figure 1e,j) were calculated based on the means of the experimental replicates. The survival of Vav-CreNfatc1^fl/fl mice as compared with littermate control mice was analyzed by Kaplan–Meier survival plot.

NFAT expression in HSPCs: (a) Expression of Nfatc1, Nfatc2, and Nfatc3 in sorted BM Lin⁻Sca1⁺c-Kit⁺ (LSK), Lin⁻Sca1⁺c-Kit⁻ (Sca1⁺), Lin⁻Sca1⁻c-Kit⁺ (c-Kit⁺), and Lin⁻Sca1⁻c-Kit⁻ (L⁻S⁻K⁻) cells from WT mice; (b) flow cytometry profiles showing distribution of BM LSK cells in WT, Nfatc2^−/−, Nfatc3^−/−, and Nfatc2^−/−Nfatc3^−/− mice. The number inside each dot plot represents percent LSK cells; (c) quantification of BM LSK cells distribution in indicated mice (WT, n = 11; Nfatc2^−/−, n = 14; Nfatc3^−/−, n = 3; and Nfatc2^−/−Nfatc3^−/−, n = 3); (d) immunofluorescence analysis for nuclear NFATc1 levels in sorted LSK, Sca1⁺, and c-Kit⁺ cells from WT mice. Co-staining with DAPI confirms nuclear NFATc1. Scale bar, 10 μm; (e) quantification of mean relative fluorescence (MRF) of nuclear NFATc1 levels in sorted BM LSK, Sca1⁺, and c-Kit⁺ cells from WT mice (LSK, n = 63; Sca1⁺, n = 73; and c-Kit⁺, n = 30 cells from three experimental replicates); (f) GFP expression representing NFATc1 levels in BM Lin⁻Sca1⁺c-Kit⁺ HSPCs from Nfatc1-eGfp-Bac tg reporter mice as compared with WT mice; (g) GFP expression representing NFATc1 levels in BM Lin⁻Sca1⁺c-Kit⁺CD150⁺ HSCs from Nfatc1-eGfp-Bac tg reporter mice as compared with WT mice; (h) GFP levels representing NFATc1 expression in BM Lin⁻IL-7R⁺Sca1⁻c-Kit⁺ CLP and Lin⁻IL-7R⁻Sca1⁻c-Kit⁺ CMP cells from Nfatc1-eGfp-Bac tg reporter mice as compared with WT mice; (i) immunofluorescence analysis for nuclear NFATc1 levels in sorted LT-HSC (Lin⁻Sca1⁺c-Kit⁺Flk2⁻) and ST-HSC (Lin⁻Sca1⁺c-Kit⁺Flk2⁺) from BM cells of WT mice. Co-staining with DAPI confirms nuclear NFATc1. Scale bar, 10 μm; (j) quantification of mean relative fluorescence (MRF) of nuclear NFATc1 levels in sorted BM LT- and ST-HSCs from WT mice. (LT-HSCs, n = 68 and ST-HSCs, n = 33 cells from three experimental replicates). Data are representative of three independent experiments, and in (c,e) (ns = not significant, one-way ANOVA) and in (j) (* p = 0.0209, unpaired t-test) are presented as mean ± s.d.