Figure 3. DAPK promotes HIF-1α degradation and DAPK deficiency increases HIF-1α levels in T cells.

(a) HIF-1α deficiency impairs Th17 differentiation. WT and Hif1a^−/− Th17 cells were re-stimulated and expression of IL-17A and IFN-γ determined. (b) Enhanced HIF-1α protein levels in Dapk^−/− Th17 cells. HIF-1α protein contents were determined in Th17 cells differentiated for 3 days (left). HIF-1α levels were quantified and normalized to tubulin (right). Mean±s.e.m., n=3. **P<0.01 for unpaired t-test. (c) DAPK-knockout does not affect HIF-1α mRNA induction during Th17 differentiation. The quantities of Hif1a transcript were determined in WT and Dapk^−/− T cells in the course of Th17 differentiation. Mean±s.e.m., n=3. (d) DAPK induces HIF-1α protein downregulation in normal T cells. Activated primary T cells were transduced with DAPK-FLAG. GFP⁺ T cells were sorted, incubated under hypoxic conditions for 48 h, and the levels of DAPK-FLAG and HIF-lα were determined. (e) DAPK reduces HIF-1α protein stability. HEK293T cells were transfected with DAPK and HIF-lα, treated with CHX (50 ng ml⁻¹) 48 h later, and the levels of HIF-1α and DAPK were measured. HIF-1α levels were quantified. (f) DAPK deficiency increases HIF-1α protein stability in T cells. WT and Dapk^−/− CD4⁺ T cells were differentiated into Th17 cells for 2 days, followed by CHX (25 ng ml⁻¹) treatment. The levels of HIF-1α protein were determined by immunoblots and were quantified. (g) DAPK-induced HIF-1α degradation is inhibited by MG132. HEK293T cells were transfected with DAPK and HIF-lα. Forty-eight hours later, cells were treated with 10 μM MG132, 200 mM NH₄Cl, 100 μg ml⁻¹ leupeptin, or 100 μM chloroquine for 6 h. The HIF-1α levels were determined. (h) DAPK-induced HIF-1α degradation is proteasome-dependent. WT and Dapk^−/− CD4⁺ T cells were differentiated into Th17 cells for 2 days, followed by MG132 (5 μM) treatment for 2 h. The levels of HIF-1α were determined. (i) DAPK deficiency increases the induction of HIF-1α protein in T cells. WT and Dapk^−/− T cells were activated with CD3/CD28 under normoxic conditions, and the levels of HIF-1α at the indicated time points were determined. Data are representative of three (a,c,e–i) or two (d) independent experiments.