Figure 1. EP1 receptor activation promoted β1-integrin expression in hepatocellular carcinoma cells.

(A). Effects of EP agonists on β1-integrin expression in Huh-7 cells. Huh-7 cells were exposed to 5 μM EP1 agonist (17-PT-PGE₂), EP2 agonist (butaprost), EP3 agonist (sulprostone) and EP4 agonist (PGE1 alcohol) for 24 h, respectively. The cropped gels are used and full-length gels are presented in Supplementary Figure S1 and S2. (B). Effects of EP antagonists on PGE₂-mediated β1-integrin expression in Huh-7 cells. Huh-7 cells were pretreated with various EP antagonists for 1 h, followed by PGE₂ for 24 h (EP1 antagonist sc19220, EP2 antagonist AH6809 and EP3 antagonist L-798106, EP4 antagonist AH23848). The cropped gels are used and full-length gels are presented in Supplementary Figure S3 and S4. (C). Effects of expression of the EP1 receptor on PGE₂-mediated β1-integrin regulation in HEK293 cells. HEK293 cells (3 × 10⁵ cells) were transfected with EP1R-pcDNA3 plasmid or empty pcDNA3 plasmid as a control. After transfection, cells expressing the EP1 receptor were selected by G418. EP1 receptor-transfected HEK293 cells were exposed to PGE₂ for 24 h, with or without sc19220 pre-treatment. Results are presented as the mean ± SD from three different experiments. *P < 0.05, compared to control cells; #P < 0.05, compared with PGE₂-treated cells. (D). RNA interference targeting the EP1 receptor suppressed PGE₂-mediated β1-integrin upregulation in Huh-7 cells. Huh-7 cells were transfected with an EP1R-siRNA. After 72 h, the cells were exposed to PGE₂ for 24 h. The cropped gels are used and full-length gels are presented in Supplementary Figure S5 and S6. Results are shown as the mean ± SD from three different experiments. ** indicates a significant difference at P < 0.01 compared with the cells without PGE₂ treatment; # indicates a significant difference at P < 0.05 compared with the siRNA negative control cells. $$ indicates a significant difference at P < 0.01 compared with the siRNA negative control cells after PGE₂ treatment. (E). Effect of anti-β1-integrin antibody on 17-PT-PGE₂-mediated cell migration in Huh-7 cells. The cell migration assay was performed in a12-well transwell. Huh-7 cells were pretreated with an anti-β1-integrin antibody for 30 min, followed by stimulation with PGE₂. The in vitro migration activity was measured after 24 h. Results are presented as the mean ± SD from three different experiments. *P < 0.05, compared with control cells; #P < 0.05, compared with 17-PT-PGE₂ –treated group. The gels have been run under the same experimental conditions.