Cross-Regulation between MST1 and BRAFV600E Figure 5. BRAFV600E affects FoxO3 mediated apoptotic gene expression. (a) 293 cells were transiently transfected with p27-promoter-luc (0.1 mg/well) and Myc-FoxO3, and co-transfected with plasmids expressing Flag-MST1 as well as plasmids expressing BRAFV600E. After 24 h, cells were harvested and analyzed by Western blotting. (b) FRO cells were transiently transfected with Myc-FoxO3 and co-transfected with plasmids expressing Flag-MST1 or BRAFV600E as indicated.Total lysates were immunoblotted with the indicated antibodies. (c) MST1-FoxO-mediated apoptosis is reversed by BRAFV600E. 293T cells were transiently transfected with Myc-FoxO3 and co-transfected with Flag-MST1 as well as BRAFV600E as indicated. After 24 hours, FoxO3 and MST-expressing cells were fixed and were then stained by the TUNEL protocol. TUNEL-positive cells (green) exhibit condensed or fragmented DAPI-stained nuclei (blue), indicative of apoptosis. Quantification of the results is shown in the right panel. MST1 expression significantly increased apoptosis, which is reduced by expression of BRAFV600E. For each experiment at least 1000 cells were counted and analyzed by Cellomics’sbioapplication. All data are presented as mean6SD. doi:10.1371/journal.pone.0016180.g005 regulate RASSF1A function via a novel mechanism. RASSF1A transfection induced p21 and p27 in a MST1-dependent manner, suggesting that the RASSF1A-MST1-FoxO3 pathway can induce cell cycle arrest. In addition, BRAFV600E suppressed the RASSF1A-MST1-mediated signal, inhibiting p21 and p27 induction, supporting the hypothesis of the present work. It has been suggested that BRAFV600E-activated ERK inhibits FoxO3 by murine double minute 2 (MDM2)-mediated degradation [25]. However, BRAFV600E did not alter HA-FoxO3 protein levels, as shown by Western blot analysis, and, interestingly, treatment with a MEK inhibitor did not abolish the BRAFV600E-mediated inactivation of FoxO3. In the present study, BRAFV600E did not induce phosphorylation of Akt/PKB and, furthermore, the addition of PI3 kinase inhibitors did not affect the inhibition of FoxO3 by BRAFV600E. Therefore, BRAFV600E did not affect FoxO3 transactivation by a PI3-Akt/PKB-dependent signaling PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e16180