Figure 6
From: PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein
PAX8 and RB co-occupy the E2F1 promoter and function antagonistically in regulating E2F1 promoter activity. (a) The effect of RB knockdown on the PAX8 transactivation of E2F1 promoter activity. At 24 h post-siRNA transfection, K1 cells were co-transfected with either vector control or pCMV-Pax8, as well as the indicated luciferase-promoter plasmid and the pCMV-β-gal plasmid. The relative luciferase units (RLUs) are normalized to β-gal activity. (b) A498 whole-cell lysate was subjected to IP using an RB antibody. IP protein complexes were analyzed using immunoblotting (IB) to detect RB and PAX8 proteins. NS indicates IP using a nonspecific antibody. (c) A498 cells were subjected to ChIP using a PAX8 antibody. IP chromatin was analyzed by ChIP–qPCR, using primers targeting the putative PAX8-binding site (Site-2). In addition, Site-1, which contains the characterized RB-E2F1-binding site, was used as a positive control. The negative control Site-C was used for comparison. Fold enrichment is shown relative to the negative control site (Site-C). Statistical significance was assessed using unpaired Student's t-test; *P<0.05. Data for a and c are the means±s.e.m. of three independent experiments for each panel. ChIP, chromatin immunoprecipitation; IP, immunoprecipitation; qPCR, quantitative PCR; RB, retinoblastoma; siRNA, small interfering RNA.
