Figure 5

Activation of FOXO3a induces Par-4 gene promoter activation. (a) DNA region showing Par-4 promoter with four consensus FOXO3a DNA binding sites spanning between +1 and −2900 bps. Below are the comparisons of various Par-4 deleted regions constructs with an ability to transactivate the Par-4 promoter region upon FOXO3a transfection or WA treatment. (b) PC-3 cells were transfected with 2.1 Kb Par-4 full length and deletion constructs (Del-1; 2.0 KB, Del-2; 1.8 KB and Del-3; 1.5 KB) promoter luciferase reporter constructs with renilla CMV as a transfection control, and/or transfected with FOXO3a expressing plasmid. Then, cell lysates were assayed for Luciferase reporter activity by showing fold induction of Par-4 activation. (c) PC-3 cells were transfected with HA-tagged FOXO3a TM or H212R mutant. The cell lysates were incubated with biotinylated DNA probes encoding WT or Mutated Par-4 promoters and pull-down assay was performed. FOXO3a was detected by immunoblotting with anti-HA to detect HA-tagged FOXO3a proteins. (d) Ch-IP analysis of FOXO3a binding to the Par-4 promoter region. PC-3 cells were transfected with FOXO3a and its genomic content was immunoprecipitated with anti-FOXO3a or control IgG. Immunoprecipitates from WT and FOXO3a-transfected cells were analyzed by quantitative PCR, using primers corresponding to Par-4 promoter