Figure 6.
Sphk2 deletion activates the HIF1α–PDK3 axis to improve metabolic fitness and function of HSCs. (A) Heatmap of metabolic genes overlapped with HIF1α targeted genes, which were upregulated in Sphk2-deficient HSCs compared with those in control HSCs. (B) Reads per kilobase per million mapped reads (RPKM) of PDK in HSCs from Sphk2Δ/Δ mice or control littermates (n = 3 mice per group). (C) Western blots of indicated proteins in HSCs from Sphk2Δ/Δ mice or control littermates. β-Actin was used as a loading control; 1#, 2#, 3#, and 4# indicated 4 individual mice. (D) Representative image (left) and quantification (right) of pPDH-E1α in HSCs from Sphk2Δ/Δ mice or control littermates (n = 100 cells from 4 mice). (E) Relative expression of PDK3 in sorted HSCs from Sphk2Δ/Δ or control mice under normoxia or hypoxia culture for 24 hours (n = 5 mice in 3 replicates). (F) ChIP-qPCR of HIF1α at the promoter region of Pdk3 in sorted HSCs from Sphk2Δ/Δ or control mice under hypoxia culture for 24 hours (n = 5 mice in 3 replicates). (G) Western blots of PDK3 in sorted HSCs from indicated mice (n = 3 replicates with 1-2 mice per replicate). (H) Western blots of PDK3 and pPDH-E1α in HSCs from indicated mice; 1# and 2# indicated 2 individual mice (n = 4 mice, 2 replicates were shown). (I) Relative intracellular pyruvate concentration, (J) lactate production, (K) LDH activity, (L) ECAR, (M) relative glucose uptake, (N) OCR, (O) intracellular ATP concentration, (P) intracellular NAD+/NADH ratio, and (Q) Cell ROX Deep (ROShigh) cells in HSCs from Pdk3Δ/+, Sphk2Δ/Δ, and Sphk2Δ/Δ; Pdk3Δ/+, or control mice, or in BM cells from 2-month-old mice as indicated (I-P: n = 5 mice in 3 replicates; Q: n = 4 mice per group). (R-T) The (R) absolute number of HSCs (n = 4 mice), (S) cell cycle analysis, and (T) annexin-V analysis of HSCs in BM from Pdk3Δ/+, Sphk2Δ/Δ, and Sphk2Δ/Δ; Pdk3Δ/+, or control mice as indicated (n = 4 mice per group). (U) PB analysis for total engrafted donor cells at the indicated number of weeks after transplantation and (V) the 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, and ∗∗∗P < .001. One-way ANOVA with Tukey's multiple comparison post hoc test, †P < .05, ††P < .01, †††P < .001. N.S., not significant.

Sphk2 deletion activates the HIF1α–PDK3 axis to improve metabolic fitness and function of HSCs. (A) Heatmap of metabolic genes overlapped with HIF1α targeted genes, which were upregulated in Sphk2-deficient HSCs compared with those in control HSCs. (B) Reads per kilobase per million mapped reads (RPKM) of PDK in HSCs from Sphk2Δ/Δ mice or control littermates (n = 3 mice per group). (C) Western blots of indicated proteins in HSCs from Sphk2Δ/Δ mice or control littermates. β-Actin was used as a loading control; 1#, 2#, 3#, and 4# indicated 4 individual mice. (D) Representative image (left) and quantification (right) of pPDH-E1α in HSCs from Sphk2Δ/Δ mice or control littermates (n = 100 cells from 4 mice). (E) Relative expression of PDK3 in sorted HSCs from Sphk2Δ/Δ or control mice under normoxia or hypoxia culture for 24 hours (n = 5 mice in 3 replicates). (F) ChIP-qPCR of HIF1α at the promoter region of Pdk3 in sorted HSCs from Sphk2Δ/Δ or control mice under hypoxia culture for 24 hours (n = 5 mice in 3 replicates). (G) Western blots of PDK3 in sorted HSCs from indicated mice (n = 3 replicates with 1-2 mice per replicate). (H) Western blots of PDK3 and pPDH-E1α in HSCs from indicated mice; 1# and 2# indicated 2 individual mice (n = 4 mice, 2 replicates were shown). (I) Relative intracellular pyruvate concentration, (J) lactate production, (K) LDH activity, (L) ECAR, (M) relative glucose uptake, (N) OCR, (O) intracellular ATP concentration, (P) intracellular NAD+/NADH ratio, and (Q) Cell ROX Deep (ROShigh) cells in HSCs from Pdk3Δ/+, Sphk2Δ/Δ, and Sphk2Δ/Δ; Pdk3Δ/+, or control mice, or in BM cells from 2-month-old mice as indicated (I-P: n = 5 mice in 3 replicates; Q: n = 4 mice per group). (R-T) The (R) absolute number of HSCs (n = 4 mice), (S) cell cycle analysis, and (T) annexin-V analysis of HSCs in BM from Pdk3Δ/+, Sphk2Δ/Δ, and Sphk2Δ/Δ; Pdk3Δ/+, or control mice as indicated (n = 4 mice per group). (U) PB analysis for total engrafted donor cells at the indicated number of weeks after transplantation and (V) the 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, and ∗∗∗P < .001. One-way ANOVA with Tukey's multiple comparison post hoc test, †P < .05, ††P < .01, †††P < .001. N.S., not significant.

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