1 mg histone H2B (Roche) in 25 mL kinase assay buffer comprised of 40 mM HEPES-NaOH (pH 7.4), 20 mM MgCl2, 1 mM DTT, phosphatase inhibitor mixture, 10 mM unlabeled ATP, and 1 mCi [c-32P]ATP. The reaction was terminated by the addition of Laemmli sample buffer and phosphorylated proteins were detected by SDS-PAGE followed by autoradiography. Apoptosis Assay Image based apoptosis assays were carried out using the APO- BrdU TUNEL Assay Kit (Invitrogen). Cells was transfected with FoxO3, MST1, and/or BRAFV600E, as indicated, and then stained with DAPI and terminal deoxynucleotidyltransferase– mediated dUTP nick end labeling (TUNEL) reagents.The proportion of apoptotic cells was determined asthe percentage of TUNEL-positive cells among all DAPI-stainedcells. Mouse Experiments and Histology The strategy and methods used for the generation of thyroid specific transgenic BRAFV600E FVB/N mice with a bovine thyroglobulin promoter have been described previously [4]. A detailed explanation of how MST1 mice are generated is described in a recent paper from our group [31]. The MST1-null mice appeared to have normal reproductive capacity when compared with wild-type mice. Tg-BRAFV600E mice were crossed with MST1-null mice to obtain Tg-BRAFV600E/MST1-null mice and Tg-BRAFV600E littermates. These mice were sacrificed at 16 weeks and their thyroids were removed after genotyping. Thyroid tissues were fixed in 10% formalin and embedded in paraffin. Five mM-thick sections were prepared and stained with H&E. Immunohistochemical analysis of paraffin embedded thyroid tissue was also performed using anti-cyclin D1 rabbit monoclonal antibody (92G2, Cell Signaling). ImageJ (http://rsbweb.nih.gov/ ij/) for microscopy was used to estimate areas without follicular architecture or colloid formation in thyroid tissues. Tg- BRAFV600E/MST1 mice (n = 10) and Tg-BRAFV600E litter- mates (n = 10) were collected and three sections of each mouse Cross-Regulation between MST1 and BRAFV600E were used for immunohistochemical and imageJ analysis. Differences in the number of undifferentiated foci and areas without follicular architecture or colloid formation between both groups were assessed by the Mann-Whitney U-test. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Chungnam National University School of Medicine (Approval ID; 2009-3-25). Statistical Analysis Results were expressed as the means 6 standard deviations. Statistical differences between two groups were analyzed by the Mann-Whitney U test. Differences were considered to be significant at P,0.05.All statistical analyses were performed using SPSS Version 16.0 for Windows (SPSS Inc., Chicago, IL). Results BRAFV600E suppresses p21 and p27 induction by RASSF1A in FRO cells To confirm that RASSF1A induces FoxO3 transactivation, RASSF1A was transiently transfected into FRO cells harboring the BRAF mutated alleles and then p21 and p27, the known transcriptional targets of FoxO3, were assessed by immunoblotting [30]. As shown in Fig 1A, endogenous RASSF1A was not detected, which is compatible with previous reports showing hypermethylation of RASSF1A in undifferentiated thyroid cancer cell lines [17,32]. Fig. 1A shows the RASSF1A-induced dose dependent increase in p21 and p27, which suggests that RASSF1A might be linked with FoxO3 transactivation. To investigate the possible crosstalk between RASSF1A and BRAFV600E, the effect of BRAFV600E on RASSF1A-mediated p21 and p27 induction was evaluated. Interestingly, BRAFWT did not affect the induction of p21 and p27 by RASSF1A, whereas BRAFV600E effectively decreased p21 and p27 (Fig. 1B), suggesting that oncogenic BRAFV600E can regulate the RASSF1A-FoxO3 signaling pathway. As BRAFV600E can inhibit FoxO3 transactivation via the ERK or Figure 1. BRAFV600E suppresses p21 and p27 induction by RASSF1A in FRO cells. (a) FRO Cells were transfected with HA-RASSF1A as indicated and incubated for 24 h. Total lysates were analyzed for p21 and p27 expression by Western blot analysis. (b) Lysates from FRO cells co- transfected with vectors for HA-RASSF1A and BRAF (wild type or the V600E mutant), as indicated, were subjected to Western blot analysis. (c) FRO cells transiently transfected with BRAFV600E and RASSF1A were incubated with MEK (U0126, 10 mM) or PI3K (wortmannin, 1 mM, Ly294002, 10 mM) inhibitors for 12 h as indicated. The lysates were subjected to western blot analysis with primary antibodies as indicated. (d) FRO cells co-transfected with RASSF1A (0.5 mg/well) and SiMST1 (20 mM/well StealthTM RNA) were incubated for 24 hrs as indicated. Abbreviations: CTL; control. doi:10.1371/journal.pone.0016180.g001 PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e16180