<p>A. Additive effect of <i>E2F-1</i> overexpression on <i>p53</i> knockdown mediated increase in <i>p73</i> promoter activity. MCF-7 cells were co-transfected with p73-PF/luciferase, pSV-β-Gal, and control or p53siRNA plasmids either in presence or in absence of <i>E2F-1</i>. Luciferase and β-galactosidase activity was determined 40 hours post-transfection. B. <i>TAp73</i> mRNA levels in MCF-7 cells transfected with control vector, p53siRNA and/or <i>E2F-1</i> cDNA. MCF-7 cells were transiently transfected with either control siRNA or <i>p53</i>-specific siRNA either in the presence or in the absence of <i>E2F-1</i>. RT-PCR analysis was performed using gene-specific primers. C & D. <i>E2F-1</i> binding is required for <i>p53</i> knockdown mediated increase in <i>p73</i> promoter activity. MCF-7 cells were co-transfected with control or p53siRNA vector and reporter construct encoding wild type or mutant <i>p73</i> promoter (p73PVUII, −220 to +71), in addition to pSV-β-Gal plasmid. The mutant PVUII promoter fragment contains mutant <i>E2F-1</i> binding sites at −155 and −132 (C). Luciferase and β-gal activity was determined 40 hours post- transfection (D). E. Occupancy of the E2F responsive element in the TAp73 promoter by E2F-1 is enhanced in MCF-7/p53siRNA cells. Detected with chromatin immunoprecipitation (ChIP) assay, DNA fragment of the TAp73 promoter was amplified from the complexes immunoprecipitated with E2F-1 antibody from the paired cell lines. Input row were the DNA fragment amplified from the extracts before immunoprecipitation. In the control immunoglobulin G (IgG) reaction, PCR was done in the eluates from beads collected after preclearing of these extracts with normal rabbit serum.</p
