Fig. 3
Involvement of PEA3 in hypoxia-induced Pref-1 expression in WI-38 cells. A WI-38 cells were transfected with control siRNA or PEA3 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 PEA3 in cell lysates. Data are presented as the mean ± S.E.M. of five experiments. *P < 0.05, compared with the control siRNA group. B WI-38 cells were subjected to hypoxia for the indicated time, and cell lysates were immunoprecipitated with an anti-PEA3 antibody; further, they were immunoblotted with antibodies for serine and PEA3. Data are presented as the mean ± S.E.M. of three experiments. *P < 0.05, compared with the control at 0 min. C Schematic of the PEA3 binding site located on the Pref-1 promoter. Cells were subjected to hypoxia (1% O2) for 30 min; the PEA3 binding site of the Pref-1 promoter region was detected through the ChIP assay. Input for for use as a positive control. Mouse polyclonal IgG for use as a negative control. Traces indicate that the three experiments produced similar results. D Cells were transfected with 0.8 μg of PEA3-Luc and 0.1 μg of pBK-CMV-Lac Z for 24 h and then subjected to hypoxia (1% O2) for another 16 h. Cells were harvested for a luciferase activity assay. Data are shown as the mean ± S.E.M., n = 3. * P < 0.05, relative to nonstimulated cells. E WI-38 cells were subjected to hypoxia (1% O2) for 30 min. In confocal microscopy, the cells were incubated with antibodies specific for PEA3, and immunoreactivity was performed through the incubation of the cells with an FITC-conjugated secondary antibody. All slides were counterstained with DAPI (blue) to distinguish the nucleus, which were visualized under an immunofluorescence microscopy (original magnification, 20 × ; n = 3)