Figure 4. NF-κB is involved in EP1 receptor-mediated β1-integrin upregulation in hepatocellular carcinoma cells.

(A). Effects of 17-PT-PGE₂ on NF-κB and IκB phosphorylation in Huh-7 cells. Huh-7 cells were treated with 5 μM 17-P-T-PGE₂ for 0, 30, 60, 120 min. Equal amounts of total proteins were separated by SDS-PAGE. Relative levels of phosphorylated and total IκBα or p65 were determined using specific antibodies. The cropped gels are used and full-length gels are presented in Supplementary Figure S8 and S9. (B). Effects of PGE₂ on NF-κB and IκB phosphorylation in EP1 receptor-expressed HEK293 cells. EP1 receptor-expressed HEK293 cells were treated with PGE₂ for 0, 30, 60, 120 min. (C). Effects of 17-PT-PGE₂ on NF-κB translocation in Huh-7 cells. Huh-7 cells were treated with 5 μM 17-PT-PGE₂ for 0, 30, 60, 120 min; the cells were then fixed with ice-cold methanol. The p65 protein was detected by immunofluorescence and activated p65 was translocated into the nuclie (arrow). All pictures were taken at 400× magnification. (D). Effect of NF-κB inhibitor on 17-PT-PGE₂-mediated β1-integrin expression in Huh-7 cells. Huh-7 cells were treated with 17-PT-PGE₂ for 24 h, with or without pre-treatment of PDTC for 24 h. Total protein was isolated and visualized with an anti-β1-integrin antibody. Levels of β-actin served as a loading control. The cropped gels are used and full-length gels are presented in Supplementary Figure S10 and S11. Densitometric quantitation of the above blots is shown. Results are presented as the mean ± SD from three different experiments. **P < 0.01, compared with control cells; ##P < 0.01, compared with 17-PT-PGE₂-treated cells. (E). Effect of NF-κB inhibitor on 17-PT-PGE₂-mediated cell migration in Huh-7 cells. The cell migration assay was performed in a 12-well transwell plate. Huh-7 cells were treated with 17-PT-PGE₂ for 12 h, with or without pre-treatment of 10 μM PDTC for 24 h. Results are presented as the mean ± SD from three different experiments. **P < 0.01, compared with control cells; ##P < 0.01, compared with 17-PT-PGE₂-treated cells. The gels have been run under the same experimental conditions.