FIG. 7.

Inhibition of the Ras/MEK/MAPK/p90Rsk and PI3K/PDK1/p90Rsk pathways results in diminished FoxM1B transcriptional activity. (A) Cartoon depicting the Ras/MEK/MAPK/p90Rsk/Myt1 and PI3K/PDK1/p90Rsk/Myt1 pathways, which prevent Myt1 phosphorylation-mediated inhibition of Cdk1 and Cdk2 activity. Also shown is the action of DN-RasN17, the MEK1/2 inhibitor U0126, the PI3K inhibitor Ly294002, DN-Akt and the Akt pharmacological kinase inhibitor, and DN-p90Rsk. (B) Cotransfection of DN-p90Rsk or DN-Ras or treatment with the MEK inhibitor U0126, the PI3K inhibitor Ly294002, or the Akt pharmacological kinase inhibitor does not influence expression of the GFP-T7-FoxM1B protein. U2OS cells were transiently transfected with CMV GFP-T7-FoxM1B plasmid with either CMV DN-p90Rsk or CMV DN-RasN17 or 50 μM U0126, 50 μM PI3K inhibitor Ly294002, or 25 μM Akt inhibitor. Protein extracts were prepared 48 h following transfection and subjected to Western blot analysis with GFP antibody. (C) Inhibition of Ras/MEK/MAPK/p90Rsk and PI3K/PDK1/p90Rsk pathways results in diminished FoxM1B transcriptional activity. U2OS-Tet^r cells were transiently cotransfected with the reporter 6×-FoxM1B-TATA-luciferase and CMV-TO-FoxM1B (500 ng) with CMV-DN-p90Rsk, CMV-DN-Ras, or DN-AKT or with 50 μM U0126 or Ly294002 either alone or together or with 25 μM Akt inhibitor. FoxM1B transcription assays in U2OS-Tet^r cells with tetracycline-inducible FoxM1B protein were performed as described in the legend to Fig. 3C and in Materials and Methods. Four separate transfection experiments were performed in triplicate to calculate standard deviations.