PDK1 activates glycolysis to raise stemness in hypoxia. (a) Immunofluorescent staining of PDK1 and HIF-1α in freezed sections (7 mm). DAPI was used as a nuclear staining. The scale bar represents 50 μm. (b) Frozen sections of central and periphery regions isolated from xenograft tumors were analyzed by H&E staining. The scale bar represents 20 μm. (c) Immunofluorescent staining of stemness-related markers (C-MYC, OCT4 and LIN28) in central and peripheral regions of xenografted tumors. DAPI is used for staining nucleus. The scale bar represents 50 μm. (d) Limiting diluted numbers of isolated tumor cells from central and peripheral regions were subcutaneously inoculated into immunodeficient mice (n=5). Serial transplantation frequency was analyzed after four weeks. (e–h) Primary cells isolated from central and peripheral regions of NTC and shPDK1 xenografted tumors were isolated. Intracellular glucose uptake (e), lactate production (f), cellular ATP levels (g) and PDH activity (h) were measured and calculated. (i) Mammosphere formation ability was analyzed by using isolated cells. The scale bar represents 100 μm. Data shown are mean±s.d. (n=3), **P<0.01 and ***P<0.001, respectively.