Figure 1. Glutamate release and transporter expression in Hep3B cells.

(A) Cells were cultured for indicated time and glutamate concentration in medium was determined and normalized to 20% O₂. ^*P < 0.05 vs 20% O₂, one-way ANOVA with Dunnett post-test. (B-C) Cells were exposed to 20% or 1% O₂ (B), or to vehicle (DMSO) or DMOG (C) for 24 h. mRNAs were analyzed by RT-qPCR and normalized to 20% O₂ or DMSO.^*P < 0.05 vs 20% O₂ or DMSO, Student's t test. (D) mRNAs were analyzed in subclones expressing shRNA directed against HIF-1α, HIF-2α or both that were exposed to 20% or 1% O₂ for 24 h. ^*P < 0.05 vs shNT at 20% O₂; ^#P < 0.05 vs shNT at 1% O₂; two-way ANOVA/Bonferroni post-test. (E) Immunoblot assays were performed using lysates from subclones exposed to 20% or 1% O₂; arrow indicates the SLC1A3-specific band. (F) SLC1A3 exons and HRE are indicated by black bars and grey oval, respectively. HRE nucleotide sequence is shown below. (G-H) Cells were exposed to 20% or 1% O₂ for 24 h. ChIP assays were performed using IgG or indicated antibody. ^*P < 0.05 vs 20% O₂, ANOVA with Bonferroni post-test. (I) Luciferase (Luc) activity was determined in cells co-transfected with pSV-Renilla and a firefly luciferase reporter containing the wild-type (WT) or mutant SLC1A3 hypoxia response element (HRE). ^*P < 0.05 vs WT at 20% O₂, ^#P < 0.05 vs WT at 1% O₂, ANOVA with Bonferroni post-test. Data are mean ± SEM or a representative blot from ≥ 3 experiments.