Figure 3. The EGFR gene contains a functional hypoxia response element.

A. Candidate HIF-binding sites (ACGTG) were identified in the Intron region between Exon 18 and Exon 19. B. A ChIP assay was performed to assess the enrichment of HIF-binding in intron 17 (no putative HIF-binding sites) and intron 18 of EGFR in BT-474 cells exposed to 20% or 1% O₂ for 4 h using IgG or antibodies against HIF-1α, HIF-2α, or HIF-1β. n=3 × N=3. Two-way ANOVA with Bonferroni post-test * P<0.05, **** P<0.001. C. Sanger sequencing of PCR-amplified genomic DNA isolated from the indicated cell lines. Reference genome sequence showing the position of T and G nucleotides variants. D-E. BT-474 (D) or 293T-cells (E) that were transiently transfected with a pGL4.23-GW -promoter construct containing a WT EGFR HRE, an HRE with a single nucleotide variant of EGFR (T>C), a fully mutated EGFR HRE or HRE from LDHA and co-transfected with a Renilla luciferase vector. Following transfection, the cells were exposed to 20% or 1% O₂ for 24 h and luciferase reporter activity (Luc) was determined. The firefly to renilla ratio was calculated and normalized by the value for 20% O_2. n=3. Student’s t-test **** P<0.0001.