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. 2004 Jul 8;23(14):2830–2840. doi: 10.1038/sj.emboj.7600279

Figure 4.

Figure 4

FoxO3 proteins mediate regulation of cyclin D2 expression by PI3-kinase. (A) Shown are immunoblots documenting the expression of cyclin D2 and Cdk2 (as a loading control) in representative clones of IκBα(A2)-, FoxO3a(WT)- and FoxO3a(A3)-expressing mouse MycER fibroblasts. Serum-starved cells were treated with 4-OHT for the indicated times. (B) Endogenous FoxO3a binds to the cyclin D2 promoter upon inhibition of PI3-kinase. Mouse MycER fibroblasts grown under the conditions as described in Figure 2A–C were analyzed by ChIP assays using the indicated antibodies. (C) Nonphosphorylatable FoxO3a (FoxO3a(A3)) blocks the loading of RNA polymerase II on the mouse cyclin D2 promoter. ChIP assays were performed with quiescent and 4-OHT-stimulated control- or FoxO3a(A3)-infected clones of mouse MycER cells using the indicated antibodies and primers for the transcription start site of mouse cyclin D2. (D) Dominant-negative FoxO enhances activation of cyclin D2 expression by Myc. Expressions of cyclin D2 and S16 as a control were analyzed by RT–PCR in pools of mouse MycER fibroblasts infected either with control virus or a retrovirus expressing a dominant-negative allele of FoxO (dnFoxO). Serum-starved cells were treated with 4-OHT for 6 h in the presence of increasing concentrations of LY294002 (5–50 μM).