Figure 4

PAX8 directly regulates E2F1 expression through stimulation of E2F1 promoter activity. The effects of PAX8 knockdown on the expression of E2F1 targets, including E2F1 itself, were analyzed at (a) the transcript and (b) the protein level. (a) Total RNA was isolated from the indicated sample at 48 h post transfection and subjected to qPCR analysis. Gene expression levels were normalized to the expression of the housekeeping genes, PPIB and YWHAZ, and presented relative to the siControl-transfected samples. The data are means±s.d. of three independent experiments. (b) Whole-cell lysates were extracted from siRNA-transfected A498, 786-O, IGROV-1 and K1 cells at 96 h post transfection. The lysates were subjected to immunoblotting using the indicated antibodies. (c) Effect of PAX8 knockdown on E2F1 promoter activity was assessed using luciferase assay. At 24 h post siRNA transfection, K1 cells were co-transfected with the indicated luciferase-promoter plasmid and pCMV-β-gal plasmid. Relative luciferase units (RLUs) are normalized to β-gal activity. (d) The promoter sequence of pE2F1-Luc was analyzed using the ConTra web tool. Site-specific primers were designed and used for ChIP–qPCR amplification for each putative PAX8-binding site (Sites-1 to -5). A region without any putative PAX8-binding site (Site-C) was used as the negative control. A498 cells were subjected to ChIP using a PAX8 antibody. IP chromatin was analyzed by ChIP–qPCR using site-specific primers. Fold enrichment is shown relative to Site-C (=1.0). Statistical significance was assessed using one-way analysis of variance; **P<0.01. The data shown in panels c and d are the means±s.e.m. of three and two independent experiments, respectively. ChIP, chromatin immunoprecipitation; IP, immunoprecipitation; qPCR, quantitative PCR; siRNA, small interfering RNA.