Fig. 5
Involvement of AP-1 in hypoxia-induced Pref-1 expression in WI-38 cells. A Cells were pretreated with curcumin (10 μM) for 30 min and then stimulated with hypoxia (1% O2) for another 4 h. Pref-1 and α-tubulin were detected in cell lysates. Data are presented as the mean ± S.E.M. for three independent experiments. *P < 0.05, compared with the hypoxia group. B WI-38 cells were transfected with control siRNA or c-Jun siRNA for 24 h and then subjected to hypoxia (1% O2) for another 4 h. Western blotting was performed to assess the levels of Pref-1, α-tubulin, and c-Jun in cell lysates. Data are presented as the mean ± S.E.M. of five experiments. *P < 0.05, compared with the control siRNA group. C WI-38 cells were exposed to hypoxia (1% O2) for 30 min. Cells were lysed with IP lysis buffer and then immunoprecipitated with the anti-c-Jun antibody. The immunoprecipitated complex was detected through immunoblotting with an anti-PEA3 antibody. Typical traces were demonstrative of three experiments. D Schematic of the AP-1 binding site located on the Pref-1 promoter. WI-38 cells were exposed to hypoxia (1% O2) for 30 min. The AP-1 binding site of the Pref-1 promoter region was detected through ChIP assay. Typical traces were presented in all three experiments. E Schematic of the PEA3 binding site located on the Pref-1 promoter. After transfection with c-Jun siRNA for 24 h, cells were subjected by hypoxia for 30 min, The PEA3 binding site of the Pref-1 promoter region was detected through ChIP assay. Typical traces were presented in all three experiments