FoxO3a negatively regulates autophagy through inhibition of FoxO1 transcription.

<p>(A) PC3 cells were transfected with siRNA for luciferase (siLuc), FoxO1 (siFoxO1), or FoxO3a (siFoxO3a), or combinations, as indicated. Cells were harvested 72 h after transfection, and cell lysates processed for immunoblot analysis of the indicated proteins. (B) FoxO3a knockdown with two FoxO3a targeting siRNA to illustrate the impact on FoxO1 protein level. (C) Real-time PCR analysis of FoxO1 and FoxO3a mRNA levels in control siRNA (gray) or FoxO3a siRNA (black) transfected cells. Cells were harvested and processed 48 h after transfection; 18S ribosomal RNA was analyzed as the normalization control. Data was presented as Mean ± S.D. (“**”, <i>p</i><0.01). (D) PC3 cells were transfected with control siRNA or FoxO3a siRNA as indicated. Following 48 h of transfection, cells were treated with either DMSO control or 10 µg/ml cycloheximide for 24 h as indicated prior to being harvested for immunoblot analysis of the indicated proteins. The FoxO1/GAPDH ratio for each condition is as presented after band quantification by ImageJ. (E) Real-time PCR analysis of endogenous FoxO1 expression level in PC3 cells transfected with either control plasmid (vector) or that expressing FoxO3a(r); in each group of the plasmid transfected cells, either control siRNA or FoxO3a siRNA were concurrently introduced. The cells are harvested for RNA preparation and q-PCR analysis 72 h post transfection; FoxO1 transcript levels were analyzed, as described in Experimental Procedures. 18S was used as the normalization control. Data was presented as Mean ± S.D. (“**”, <i>p</i><0.01). All experiments have been performed three times with similar results.