Sphk2 deletion increases hypoxic response and metabolic fitness in HSCs. (A) Enriched pathways of differentially expressed genes in HSCs from Sphk2^Δ/Δ or control littermates. The enriched P value is derived from Fisher's exact test. (B) Signature enrichment plots from GSEA using glycolysis and oxidative phosphorylation gene sets in HSCs from WT control mice or Sphk2^Δ/Δ mice. (C-K) (C) Relative glucose uptake, (D) relative intracellular pyruvate concentration, (E) lactate production, (F) lactate dehydrogenase (LDH) activity, (G) extracellular acidification rate (ECAR), (H) oxygen consumption rate (OCR), (I) intracellular ATP concentration, (J) intracellular NAD⁺/NADH ratio, and (K) Cell ROX Deep (ROS^high) cells in HSCs from Sphk2^Δ/Δ or control mice (C-J: n = 5 mice in 3 replicates; K: n = 5 mice). (L) Relative HIF activity in HSCs from Sphk2^Δ/Δ or control mice. The relative expression values of 64 differentially expressed HIF targeted genes are presented. Each dot represents the mean level of 3 replicates (n = 3 mice per genotype). (M) Relative expression of hypoxia response genes in HSCs from Sphk2^Δ/Δ or control mice under hypoxia or normoxia culture for 24 hours (n = 5 mice in 3 replicates). (N-P) (N) Western blots, (O) quantification of HIF1α and Sphk2 protein expression, and (P) relative expression of hypoxia response genes in sorted HSCs from indicated mice under hypoxia culture for 24 hours (n = 5 mice in 3 replicates). (Q-Y) (Q) Relative glucose uptake, (R) relative intracellular pyruvate concentration, (S) lactate production, (T) LDH activity, (U) ECAR, (V) OCR, (W) intracellular ATP concentration, (X) intracellular NAD⁺/NADH ratio, and (Y) Cell ROX Deep (ROS^high) cells in HSCs from indicated mice (Q-X: n = 5 mice in 3 replicates; Y: n = 5 mice). (Z-AA) The (Z) absolute number and (AA) cell cycle of HSCs from indicated mice (n = 5 mice per group). (AB-AC) One hundred purified HSCs from indicated donor mice were transplanted into irradiated mice with 2 × 10⁵ recipient BM cells. (AB) PB analysis for total engrafted donor cells at the indicated number of weeks after transplantation and (AC) percentage of donor-derived B, T, and myeloid lineage cells at 16 weeks after transplantation (n = 5 mice per group). 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.