Figure 5.
Sphk2 bridges the association between PHD2 and VHL to repress HIF1α activity in HSCs. (A) Western blots of Sphk2 and HIF1α in HSCs with Sphk2 overexpression (OE) or knockdown (KD) as indicated under hypoxia (quantified in supplemental Figure 5B). (B) Western blots of the HIF1α, HIF2α, VHL, PHD2, and Sphk2 in sorted HSCs from Sphk2Δ/Δ or control mice under hypoxia culture for 24 hours. β-Actin was used as a loading control (n = 4 mice, replicates from 2 individual mice were shown and were quantified in supplemental Figure 5C). (C) Western blots of HIF1α concentrations in HSCs purified from Sphk2Δ/Δ or control mice at indicated time after cycloheximide (CHX) treatment under hypoxia. (D) Western blots of HIF1α concentrations in control HSCs or HSCs with Sphk2 overexpression with or without proteasome inhibitor MG132 or lysosome inhibitor chloroquine (CQ) treatment under hypoxia culture for 24 hours. β-Actin was used as a loading control. (E) Western blots of Ub and VHL in the input and immunoprecipitate with anti-HIF1α antibody in HSCs from Sphk2Δ/Δ or control mice under hypoxia culture for 24 hours. (F) Representation of Sphk2–VHL–PHD2 interacting surfaces predicted by AlphaFold2 and GRAMM-X. (G) Western blots of HIF1α, Sphk2, PHD2, and VHL in the input and immunoprecipitate with anti-Sphk2 antibody in sorted HSCs under normoxia and hypoxia culture for 24 hours. (H) Western blots of HIF1α, VHL, and PHD2 in the input and immunoprecipitate with anti-VHL antibody in HSCs from Sphk2Δ/Δ or control mice under hypoxia culture for 24 hours. (I) The interaction of VHL-V5 (upper) or PHD2-V5 (lower) with Sphk2-GST, Sphk2-D1-GST, Sphk2-D2-GST, and Sphk2-D3-GST in GST-pulldown assay. (J) Western blots of PHD2, VHL, and Sphk2-Flag in the input and immunoprecipitate with anti-Flag antibody in 293T cells under hypoxia. (K-L) (K) Representative staining images and (L) quantification of the proximity ligation assays (PLAs) of indicated proteins in HSCs under hypoxia culture for 24 hours (n = 25 cells from 3 mice). (M) Western blots of HIF1α, VHL, PHD2, and Sphk2 in cytoplasmic and nuclear-sorted HSCs from Sphk2Δ/Δ or control mice under hypoxia culture for 24 hours. (N) Representative images (left) and quantification (right) of VHL, PHD2, and HIF1α in sorted HSCs from Sphk2Δ/Δ or control mice under hypoxia culture for 24 hours (n = 25 cells from 3 mice). (O) Western blots of Ub-HA, HIF1α-V5, and VHL in the input and immunoprecipitate with anti-V5 antibody in 293T cells. (P) Western blots of the HIF1α-HA, VHL, PHD2, and Sphk2-Flag in the nucleus (Nuc) and cytoplasm (Cyto) after overexpression of Sphk2 or Sphk2 mutants as indicated under hypoxia. (Q) Illustration of Sphk2-PHD2-VHL complex. (R) Experimental strategy. (S) Western blots of HIF1α and Sphk2 in HSCs from Sphk2Δ/Δ or control mice after overexpression of Sphk2 or Sphk2 mutants as indicated under hypoxia culture for 24 hours (quantified in supplemental Figure 5P). (T) PB analysis for total engrafted donor cells at the indicated number of weeks after transplantation (n = 5 mice per group). (K,N) Scale bars, 5 μm. Data represented as mean ± standard deviation. Two-tailed Student t tests were used to assess statistical significance. ∗∗∗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 bridges the association between PHD2 and VHL to repress HIF1α activity in HSCs. (A) Western blots of Sphk2 and HIF1α in HSCs with Sphk2 overexpression (OE) or knockdown (KD) as indicated under hypoxia (quantified in supplemental Figure 5B). (B) Western blots of the HIF1α, HIF2α, VHL, PHD2, and Sphk2 in sorted HSCs from Sphk2Δ/Δ or control mice under hypoxia culture for 24 hours. β-Actin was used as a loading control (n = 4 mice, replicates from 2 individual mice were shown and were quantified in supplemental Figure 5C). (C) Western blots of HIF1α concentrations in HSCs purified from Sphk2Δ/Δ or control mice at indicated time after cycloheximide (CHX) treatment under hypoxia. (D) Western blots of HIF1α concentrations in control HSCs or HSCs with Sphk2 overexpression with or without proteasome inhibitor MG132 or lysosome inhibitor chloroquine (CQ) treatment under hypoxia culture for 24 hours. β-Actin was used as a loading control. (E) Western blots of Ub and VHL in the input and immunoprecipitate with anti-HIF1α antibody in HSCs from Sphk2Δ/Δ or control mice under hypoxia culture for 24 hours. (F) Representation of Sphk2–VHL–PHD2 interacting surfaces predicted by AlphaFold2 and GRAMM-X. (G) Western blots of HIF1α, Sphk2, PHD2, and VHL in the input and immunoprecipitate with anti-Sphk2 antibody in sorted HSCs under normoxia and hypoxia culture for 24 hours. (H) Western blots of HIF1α, VHL, and PHD2 in the input and immunoprecipitate with anti-VHL antibody in HSCs from Sphk2Δ/Δ or control mice under hypoxia culture for 24 hours. (I) The interaction of VHL-V5 (upper) or PHD2-V5 (lower) with Sphk2-GST, Sphk2-D1-GST, Sphk2-D2-GST, and Sphk2-D3-GST in GST-pulldown assay. (J) Western blots of PHD2, VHL, and Sphk2-Flag in the input and immunoprecipitate with anti-Flag antibody in 293T cells under hypoxia. (K-L) (K) Representative staining images and (L) quantification of the proximity ligation assays (PLAs) of indicated proteins in HSCs under hypoxia culture for 24 hours (n = 25 cells from 3 mice). (M) Western blots of HIF1α, VHL, PHD2, and Sphk2 in cytoplasmic and nuclear-sorted HSCs from Sphk2Δ/Δ or control mice under hypoxia culture for 24 hours. (N) Representative images (left) and quantification (right) of VHL, PHD2, and HIF1α in sorted HSCs from Sphk2Δ/Δ or control mice under hypoxia culture for 24 hours (n = 25 cells from 3 mice). (O) Western blots of Ub-HA, HIF1α-V5, and VHL in the input and immunoprecipitate with anti-V5 antibody in 293T cells. (P) Western blots of the HIF1α-HA, VHL, PHD2, and Sphk2-Flag in the nucleus (Nuc) and cytoplasm (Cyto) after overexpression of Sphk2 or Sphk2 mutants as indicated under hypoxia. (Q) Illustration of Sphk2-PHD2-VHL complex. (R) Experimental strategy. (S) Western blots of HIF1α and Sphk2 in HSCs from Sphk2Δ/Δ or control mice after overexpression of Sphk2 or Sphk2 mutants as indicated under hypoxia culture for 24 hours (quantified in supplemental Figure 5P). (T) PB analysis for total engrafted donor cells at the indicated number of weeks after transplantation (n = 5 mice per group). (K,N) Scale bars, 5 μm. Data represented as mean ± standard deviation. Two-tailed Student t tests were used to assess statistical significance. ∗∗∗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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