Figure 1.
Sphk2 deletion increases HSC function during homeostasis. (A-C) (A) The absolute number of HSPCs (LT-HSC, ST-HSC, MPP, and SLAM HSC) (WT n = 6 mice, Sphk1Δ/Δ n = 6 mice, and Sphk2Δ/Δ n = 5 mice) in the BM, (B) cell cycle analysis (WT n = 4 mice, Sphk1Δ/Δ n = 8 mice, and Sphk2Δ/Δ n = 4 mice), and (C) apoptosis analysis (n = 5 mice per group) of HSCs in BM from Sphk1Δ/Δ, Sphk2Δ/Δ, or control mice. (D) Western blots (top) and quantification (bottom left) of relative Sphk2 protein expression and Sphk2 mRNA expression (bottom right) in each hematopoietic population as indicated from wild-type C57BL/6J mice (n = 3 mice). (E) Targeted metabolomics detection of S1P via LC/MS in HSCs (n = 3 mice). (F) Representative images of sorted HSCs immunostained with Sphk2 (left) and quantification of Sphk2 expression (right) in the cytoplasm (Cyto-Sphk2) and nuclear (Nuc-Sphk2) in HSCs from Sphk2Δ/Δ or control mice (n = 25 cells from 3 mice). (G) Scheme for quantification of functional HSCs by transplantation assay. A total of 2 × 105 BM cells from Sphk1Δ/Δ, Sphk2Δ/Δ, or control mice were transplanted into irradiated mice along with 2 × 105 recipient BM cells. A total of 1 × 106 BM cells from primary or secondary recipient mice were transplanted into irradiated mice in secondary transplantation or tertiary transplantation, respectively. (H) PB analysis for total engrafted donor cells at the indicated number of weeks after transplantation and (I) percentage of donor-derived B, T, and myeloid lineage cells at 16 weeks after transplantation (1° WT n = 9-10 mice, Sphk1Δ/Δ n = 8-10 mice, and Sphk2Δ/Δ n = 7-8 mice; 2° WT n = 9 mice, Sphk1Δ/Δ n = 6-7 mice, and Sphk2Δ/Δ n = 6-7 mice; 3° WT n = 4-7 mice, Sphk1Δ/Δ n = 5-8 mice, and Sphk2Δ/Δ n = 4-6 mice). (J) Scheme for quantification of HSC self-renewal potential by transplantation assay. A total of 100 purified HSCs from Sphk2Δ/Δ or control mice were transplanted into irradiated mice along with 2 × 105 recipient BM cells. A total of 1 × 106 BM cells from primary recipient mice were transplanted into irradiated mice in secondary transplantation. (K) PB analysis for total engrafted donor cells at the indicated number of weeks after transplantation and (L) percentage of donor-derived B, T, and myeloid lineage cells at 16 weeks after transplantation (1° WT n = 7-8 mice, Sphk2Δ/Δ n = 4-6 mice; 2° WT n = 6-8 mice, and Sphk2Δ/Δ n = 5-8 mice). 1°, primary transplantation; 2°, secondary transplantation; 3°, tertiary transplantation. (F) Scale bar 5 μm. Data represented as mean ± standard deviation. Two-tailed Student t test assessed statistical significance, ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. One-way ANOVA with Tukey‘s multiple comparison post hoc test, †P < .05, ††P < .01, and †††P < .001. N.S., not significant.

Sphk2 deletion increases HSC function during homeostasis. (A-C) (A) The absolute number of HSPCs (LT-HSC, ST-HSC, MPP, and SLAM HSC) (WT n = 6 mice, Sphk1Δ/Δ n = 6 mice, and Sphk2Δ/Δ n = 5 mice) in the BM, (B) cell cycle analysis (WT n = 4 mice, Sphk1Δ/Δ n = 8 mice, and Sphk2Δ/Δ n = 4 mice), and (C) apoptosis analysis (n = 5 mice per group) of HSCs in BM from Sphk1Δ/Δ, Sphk2Δ/Δ, or control mice. (D) Western blots (top) and quantification (bottom left) of relative Sphk2 protein expression and Sphk2 mRNA expression (bottom right) in each hematopoietic population as indicated from wild-type C57BL/6J mice (n = 3 mice). (E) Targeted metabolomics detection of S1P via LC/MS in HSCs (n = 3 mice). (F) Representative images of sorted HSCs immunostained with Sphk2 (left) and quantification of Sphk2 expression (right) in the cytoplasm (Cyto-Sphk2) and nuclear (Nuc-Sphk2) in HSCs from Sphk2Δ/Δ or control mice (n = 25 cells from 3 mice). (G) Scheme for quantification of functional HSCs by transplantation assay. A total of 2 × 105 BM cells from Sphk1Δ/Δ, Sphk2Δ/Δ, or control mice were transplanted into irradiated mice along with 2 × 105 recipient BM cells. A total of 1 × 106 BM cells from primary or secondary recipient mice were transplanted into irradiated mice in secondary transplantation or tertiary transplantation, respectively. (H) PB analysis for total engrafted donor cells at the indicated number of weeks after transplantation and (I) percentage of donor-derived B, T, and myeloid lineage cells at 16 weeks after transplantation (1° WT n = 9-10 mice, Sphk1Δ/Δ n = 8-10 mice, and Sphk2Δ/Δ n = 7-8 mice; 2° WT n = 9 mice, Sphk1Δ/Δ n = 6-7 mice, and Sphk2Δ/Δ n = 6-7 mice; 3° WT n = 4-7 mice, Sphk1Δ/Δ n = 5-8 mice, and Sphk2Δ/Δ n = 4-6 mice). (J) Scheme for quantification of HSC self-renewal potential by transplantation assay. A total of 100 purified HSCs from Sphk2Δ/Δ or control mice were transplanted into irradiated mice along with 2 × 105 recipient BM cells. A total of 1 × 106 BM cells from primary recipient mice were transplanted into irradiated mice in secondary transplantation. (K) PB analysis for total engrafted donor cells at the indicated number of weeks after transplantation and (L) percentage of donor-derived B, T, and myeloid lineage cells at 16 weeks after transplantation (1° WT n = 7-8 mice, Sphk2Δ/Δ n = 4-6 mice; 2° WT n = 6-8 mice, and Sphk2Δ/Δ n = 5-8 mice). 1°, primary transplantation; 2°, secondary transplantation; 3°, tertiary transplantation. (F) Scale bar 5 μm. Data represented as mean ± standard deviation. Two-tailed Student t test assessed statistical significance, ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. One-way ANOVA with Tukey‘s multiple comparison post hoc test, †P < .05, ††P < .01, and †††P < .001. N.S., not significant.

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