Figure 4. Cytoplasmic presence of HIF-1α and co-localization with DAPK in T cells.

(a) Predominant nuclear location of HIF-1α in HeLa cells. HeLa cells were cultured in normoxic or hypoxic (1% O₂) conditions for 24 h. Nuclear (N) and cytoplasmic (C) fractions were isolated, and HIF-1α expression levels were determined. HDAC2 and GAPDH were used as the nuclear marker and cytosolic marker, respectively. (b,c) Cytoplasmic presence of HIF-1α in T cells. WT T cells were activated with anti-CD3/CD28 for the indicated times (b), while naive CD4⁺ T cells were differentiated into Th17 for 48 h (c). Nuclear and cytoplasmic fractions were prepared, and HIF-1α expressions were examined. (d) Image analysis of HIF-1α localization in Th17 and HeLa cells. Th17 cells (c) and HeLa cells (a) were stained with anti-HIF-1α and DAPI and analysed by confocal microscopy. Scale bar, 10 μm. (e) Co-localization of HIF-1α and DAPK in Jurkat cells. DAPK-FLAG-overexpressing Jurkat cells were cultured under hypoxic (1% O₂) conditions for 4 h, fixed and stained with anti-FLAG, anti-HIF-1α and DAPI, and analysed by confocal microscopy. Scale bar, 5 μm. (f) Co-localization of HIF-1α and DAPK in Th17 and Th0 cells. WT naive T cells were differentiated into Th17 (top) or Th0 cells (bottom) for 2 days, fixed and stained with anti-HIF-1α, anti-DAPK and DAPI, and analysed by confocal microscopy. Scale bar, 5 μm. (g) Interaction between HIF-1α and DAPK. HEK293T cells were transfected with DAPK-FLAG or HIF-1α, as indicated. The whole-cell lysates (WCL) were immunoprecipitated with either anti-FLAG (top) or anti-HIF-1α (bottom). The contents of DAPK-FLAG and HIF-1α in the precipitates and lysates were determined. (h) Involvement of the DAPK cytoskeleton domain in HIF-1α binding. HEK293T cells were transfected with HIF-1α-HA, and WT DAPK-FLAG or DAPK(ΔCyto)-FLAG. The WCL were immunoprecipitated with anti-HA, and the amounts of DAPK and HIF-1α were determined. (i) Association of DAPK with the endogenous HIF-1α in hypoxic T cells. DAPK in normoxic and hypoxic (1% O₂ for 4 h) Jurkat cells was precipitated by anti-DAPK, and amounts of the associated HIF-1α were determined by western blot. Data are representative of three (a–h) or two (i) independent experiments.