FIGURE 2.

Tumor hypoxia induces CXC chemokine ligand 14 (CXCL14) expression through hypoxia‐inducible factor‐1α (HIF‐1α) and HIF‐2α. (A) Time course of CXCL14 mRNA expression after exposure of GBM8901 cells to <1% O₂. (B–E) Levels of (B) CXCL14 mRNA and (C,D) cellular protein in human glioblastoma cells (U87, U251, and GBM8901) and primary glioblastoma cells (GBM04T and GBM09T) or (E) its secreted protein in medium at 24 h after normoxic (N.) and hypoxic (H.) treatment (<1% O₂). (F) Protein levels of CXCL14, HIF‐1α, and HIF‐2α in U251 and GBM8901 cells with or without HIF‐1α or HIF‐2α knockdown at 24 h after hypoxic treatment (<1% O₂). (G) Secreted CXCL14 protein in GBM04T and GBM09T glioblastoma cells with or without HIF‐1α (LW6) or HIF‐2α (PT2399) inhibitor at 24 h after hypoxic treatment (<1% O₂). (H) One putative hypoxia response element (HRE) was identified in human CXCL14 promoter. (I) Luciferase reporter plasmids carrying the WT or mutant CXCL14 promoter regions were cotransfected with the Renilla luciferase reporter plasmid into U251, GBM8901, GBM04T, and GBM09T cells; the cells were treated with or without hypoxia (<1% O₂) for 24 h. (J) ChIP followed by real‐time PCR (ChIP‐qPCR) assay of HIF‐1α or HIF‐2 α binding in CXCL14 promoter in response to hypoxia (<1% O₂) for 24 h. Results are expressed as percentage of input. Error bars, SD within triplicate experiments. ***p < 0.001; ****p < 0.0001, compared with normoxia.. ^## p < 0.01, compared with hypoxia + vehicle group