Figure 3.
Sphk2 deletion prevents the accumulation of HSC aging phenotypes. (A) Western blots (left) and quantification analyses (right) of Sphk2 expression in yHSCs and oHSCs. β-Actin was used as a loading control; 1# and 2# indicated 2 individual mice (n = 4 mice, 2 replicates were presented, and 4 replicates were quantified). (B) Western blots (left) and quantification analyses (right) of Sphk2 expression in the HSC subpopulations from 22- to 24-month-old mice as indicated. β-Actin was used as a loading control (n = 1-2 mice per replicate). (C) The absolute number of HSC subpopulations from 22- to 24-month-old Sphk2Δ/Δ or control mice (n = 7 mice per group). (D–F) The frequency of (D) cell cycle, (E) γH2AX+ cells, and (F) Cell ROX Deep (ROShigh) cells in HSCs from 22- to 24-month-old Sphk2Δ/Δ or control mice (WT n = 8 mice and Sphk2Δ/Δ n = 7 mice). (G) PB analysis for the percentage of B, T, and myeloid lineage cells from young or old Sphk2Δ/Δ or control mice as indicated (2-month-old WT n = 5 mice, 22- to 24-month-old WT n = 8 mice, and 22- to 24-month-old Sphk2Δ/Δ n = 7 mice). (H) Scheme for quantification of functional HSCs by transplantation assay. A total of 2 × 105 BM cells from 22- to 24-month-old 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. (I,K) PB analysis for total engrafted donor cells at the indicated number of weeks after transplantation and (J,L) the percentage of donor-derived B, T, and myeloid lineage cells at 16 weeks after transplantation (primary transplantation n = 6-8 mice per group, secondary transplantation WT n = 3-4 mice, and Sphk2Δ/Δ n = 5-6 mice). 1°, primary transplantation; 2°, secondary transplantation. (M) Experimental strategy and (N) Western blots for Sphk2 overexpression in yHSCs. (O) PB analysis for total engrafted donor cells at the indicated number of weeks after transplantation and (P) the percentage of donor-derived B, T, and myeloid lineage cells at 16 weeks after transplantation (vector n = 5 mice and Sphk2 OE n = 5 mice). Overexpression (OE). (Q) t-SNE plot depicting the distribution of yHSCs, oHSCs from WT mice, or oHSCs from Sphk2Δ/Δ mice (oHSC Sphk2Δ/Δ) as indicated. (R) Heatmap, (S) gene expression score, and (T) signature enrichment plots from GSEA for age-repressed, age-induced, lymphoid, CLP, myeloid, and HSC stemness genes in yHSCs, oHSCs, and Sphk2Δ/Δ oHSCs. yHSCs indicate SLAM HSCs obtained from 2-month-old mice. oHSCs indicate SLAM HSCs obtained from 22- to 24-month-old mice. Data represented as mean ± standard deviation. Two-tailed Student t tests were used to assess 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 prevents the accumulation of HSC aging phenotypes. (A) Western blots (left) and quantification analyses (right) of Sphk2 expression in yHSCs and oHSCs. β-Actin was used as a loading control; 1# and 2# indicated 2 individual mice (n = 4 mice, 2 replicates were presented, and 4 replicates were quantified). (B) Western blots (left) and quantification analyses (right) of Sphk2 expression in the HSC subpopulations from 22- to 24-month-old mice as indicated. β-Actin was used as a loading control (n = 1-2 mice per replicate). (C) The absolute number of HSC subpopulations from 22- to 24-month-old Sphk2Δ/Δ or control mice (n = 7 mice per group). (D–F) The frequency of (D) cell cycle, (E) γH2AX+ cells, and (F) Cell ROX Deep (ROShigh) cells in HSCs from 22- to 24-month-old Sphk2Δ/Δ or control mice (WT n = 8 mice and Sphk2Δ/Δ n = 7 mice). (G) PB analysis for the percentage of B, T, and myeloid lineage cells from young or old Sphk2Δ/Δ or control mice as indicated (2-month-old WT n = 5 mice, 22- to 24-month-old WT n = 8 mice, and 22- to 24-month-old Sphk2Δ/Δ n = 7 mice). (H) Scheme for quantification of functional HSCs by transplantation assay. A total of 2 × 105 BM cells from 22- to 24-month-old 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. (I,K) PB analysis for total engrafted donor cells at the indicated number of weeks after transplantation and (J,L) the percentage of donor-derived B, T, and myeloid lineage cells at 16 weeks after transplantation (primary transplantation n = 6-8 mice per group, secondary transplantation WT n = 3-4 mice, and Sphk2Δ/Δ n = 5-6 mice). 1°, primary transplantation; 2°, secondary transplantation. (M) Experimental strategy and (N) Western blots for Sphk2 overexpression in yHSCs. (O) PB analysis for total engrafted donor cells at the indicated number of weeks after transplantation and (P) the percentage of donor-derived B, T, and myeloid lineage cells at 16 weeks after transplantation (vector n = 5 mice and Sphk2 OE n = 5 mice). Overexpression (OE). (Q) t-SNE plot depicting the distribution of yHSCs, oHSCs from WT mice, or oHSCs from Sphk2Δ/Δ mice (oHSC Sphk2Δ/Δ) as indicated. (R) Heatmap, (S) gene expression score, and (T) signature enrichment plots from GSEA for age-repressed, age-induced, lymphoid, CLP, myeloid, and HSC stemness genes in yHSCs, oHSCs, and Sphk2Δ/Δ oHSCs. yHSCs indicate SLAM HSCs obtained from 2-month-old mice. oHSCs indicate SLAM HSCs obtained from 22- to 24-month-old mice. Data represented as mean ± standard deviation. Two-tailed Student t tests were used to assess 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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