Figure 1.

EP4 receptor promoter activities of 5′ deletion mutants and point mutated HRE in HCA‐7 cells. HCA‐7 cells were cultured under low (Low; 2 × 10⁵ cells/each well of 6‐well plate) or high (High; 2 × 10⁶ cells/each well of 6‐well plate) cellular density conditions, and were then transfected with reporter gene plasmids containing WT or each promoter‐mutated plasmid and subjected to the EP4 receptor promoter luciferase assay, as described in the Materials & Methods. (A) The promoter deletion maps and corresponding luciferase activities of each WT (−1238/+1) or the 5′ deletion of EP4 promoter‐containing reporter gene plasmids (del 1 to del 4) obtained from HCA‐7 cells cultured under low or high cellular density conditions. (B, C, D) The luciferase activities of HCA‐7 cells cultured under low cellular density conditions transfected with WT (−1238/+1) (B), del 3 (−197/+1) (C), or del 4 (−160/+1) (D) of each human EP4 receptor promoter plasmid concomitantly with either HA‐control vector plasmids or HA‐tagged HIF‐1α expression plasmids. (E) Schematic maps of WT (−1238/+1) or HRE point mutated WT (mut‐HRE (−1238/+1)) of the EP4 promoter. (F) ChIP assay with anti‐HIF‐1α antibodies performed to verify the binding of HIF‐1α to WT (−1238/+1) or HRE point mutated WT (mut‐HRE (−1238/+1)) of the EP4 promoter in HCA‐7 cells cultured under high cellular density conditions. (G) The luciferase activities of each WT (−1238/+1) or HRE point mutated WT (mut‐HRE (−1238/+1)) of EP4 promoter‐containing reporter gene plasmids obtained from HCA‐7 cells cultured under low or high cellular density conditions. (H) The luciferase activities of HCA‐7 cells cultured under low cellular density conditions transfected either with WT (−1238/+1) or HRE point mutated WT (mut‐HRE (−1238/+1)) of EP4 promoter‐containing reporter gene plasmids concomitantly with either HA‐control vector plasmids or HA‐tagged HIF‐1α expression plasmids. Luciferase activity was assessed, as described in the Materials & Methods. Data are normalized to low cellular density‐cultured cells transfected with WT, or HA control vector plasmid‐transfected cells under low cellular density conditions as 100%. Data are the mean ± SD of three or more than three independent experiments. *P < 0.05, t test or one‐sample t test, significantly different from low cellular density‐cultured cells transfected with WT or mutated human EP4 receptor promoter plasmids. ^† P < 0.05, t test or one‐sample t test, significantly different from HA control vector plasmid‐transfected cells under low cellular density conditions. n.s.; not significant