Figure 3.

Par-4 is a direct target of Foxo3a. A – C, Time-course of Par-4 mRNA upregulation following 4-OH Tamoxifen treatment in BT-474 (A), SKBR3 (B), or MCF-7 (C) cells stably expressing Foxo3a TM-ER fusion. D, Western analysis showing Par-4 upregulation 24 hours after 4-OHT treatment. E, Schematic of the Par-4 promoter surrounding the transcriptional start site. Eight regions (~500bp each) were cloned upstream of luciferase for use in reporter gene experiments. Putative Foxo3a binding sites in region 2, 6, and 8 are shown. F, 293T cells were transfected with luciferase constructs containing each promoter region together with empty vector or constitutively active Foxo3a TM, and luciferase expression was measured 24 hours later. pGL3: empty vector (negative control); FHRE: forkhead response element (positive control) G, Chromatin immunoprecipitation (ChIP) analysis of Foxo3a occupancy at indicated regions of the Par-4 promoter in BT-474 cells treated with vehicle or MK-2206 for 24 hours. A distal region (-10 kb) of the Par-4 promoter was used as a negative control, and the Puma promoter was used as a positive control. Data are expressed as fold-enrichment over IgG IP. Significance was determined by Student’s t-test and data are presented as mean plus SD. **, p<0.01; ***, p<0.001.