Figure 5.

FOXO3a binds to the promoter of PUMA and positively regulates its expression. (A) FOXO3a is not able to directly bind to the promoters of the genes involved in autophagic signaling. MG63-R12 and MG63-R12+FOXO3a cells were subjected to a ChIP assay using the FOXO3a antibody, and the binding of autophagic signaling genes, including LC3, SIRT1, Beclin-1, ATG5 and ATG7, was assessed by qRT-PCR. The expression levels of each gene were normalized against the control vector. (B) FOXO3a specifically binds to the promoter of PUMA. The same DNA in (A) was used to examine the binding of apoptotic signaling genes, including p53, PUMA, Bax, Caspase-3 and Apaf-1. (C) A schematic diagram of the PUMA promoter. The promoter region (-1 - -1500) of PUMA is indicated, and the consensus sequence of the FOXO3a binding site is indicated in red. The mutant FOXO3a binding site is indicated in blue. (D) Luciferase assay. MG63-R12, MG63-R12+FOXO3a, U2OS-R5, and U2OS-R5+FOXO3a cells were co-transfected the firefly luciferase reporter vector pGL4.26-P_PUMA-WT-Luc or pGL4.26-P_PUMA-Mutant-Luc with the Renilla luciferase vector pRL-TK, respectively. After incubation at 37°C for 24 hrs, the cells were subjected to a luciferase assay using the Dual Luciferase Reporter Assay System. The luciferase activities in MG63-R12+FOXO3a and U2OS-R5+FOXO3a cells were normalized against MG63-R12 and U2OS-R5 cells, respectively (**P < 0.001).