Figure 3.

Receptor-interacting protein 1 suppresses the p27^Kip1 promoter by inhibiting the forkhead transcription factors. (A) RIP1 inhibits the p27^Kip1 promoter in reporter assays in a dose-dependent manner. 293 cells were transfected with wild-type RIP1 along with p27^Kip1-Luc (p27-Luc). Luciferase activity was measured after 24 h. (B) 293 cells were transfected with wild-type RIP1 or mutant RIP1 along with p27^Kip1-Luc. The RIP1 mutants used here are deletions of the kinase domain (RIP1-DKD), intermediate domain (RIP1-DID) or death domain (RIP1-DDD). (C) RIP1 inhibits the activity of forkhead transcription factors in reporter assays. An FHRE-Luc (pGL3-E4-DBEx6) plasmid was co-transfected into 293 cells with either RIP1 or empty vector and luciferase activity was measured after 24 h. (D) The effect of RIP1 on the p27^Kip1 promoter is rescued by either wild–type FoxO3a or a constitutively active (AAA) mutant FoxO3a. The experiment was conducted in 293 cells by transfecting cells with RIP1 plus p27^Kip1-Luc and either empty vector or wild-type or mutant FoxO3a. (E) Mutation of a FoxO response element in the p27^Kip1-Luc promoter abolishes the ability of RIP1 to suppress the p27^Kip1 promoter. In this experiment, RIP1 was co-transfected into 293 cells with either wild-type or a mutant p27^Kip1-Luc promoter followed by luciferase assays. In all reporter assays, Renilla luciferase activity was measured as an internal control. Three independent experiments were performed in triplicate and a representative experiment is shown. Expression of RIP1 was confirmed by western blot (data not shown). RIP1, receptor-interacting protein 1; RLU, RLU, relative luciferase unit.