expression was calculated using untreated cells and KAT4C baseline values as a reference, or as indicated otherwise. Cell cycle and apoptosis assessment Cells were plated at 16105 cells per well in 6-well plates in 2 mL media overnight. BEZ235 or vehicle were added at indicated doses for 24 hours (KAT4C, 8505C, BHP7-13 and WRO82-1) or 72 hours (TT), adherent cells were trypsinized, washed with PBS, fixed with cold 70% ethanol and incubated with RNase A (100 mg/mL; Sigma) and propidium iodide (5 mg/mL; Sigma) at 37uC for 15 min. Cell cycle distribution was assessed by DNA content detected by flow cytometry (BD FACScalibur Flow Cytometer, BD Biosciences). Cells were plated at 26104 cells per well in 6-well plates in 2 mL media overnight and treated with BEZ235 for 96 hours. Floating cells and trypsinized adherent cells were collected and samples were prepared as described above. Apoptotic sub-G1 cells were detected by DNA content by flow cytometry. Each condition was performed in triplicate. Flank xenograft tumor therapy Eight-week-old athymic female nude mice (National Laboratory Animal Center, Taiwan) were anesthetized with intraperitoneal injection of ketamine hydrochloride (90 mg/kg; Nang Kuang Pharmaceutical Co.) and xylazine hydrochloride (9 mg/kg; Bayer) before implantation of thyroid cancer cells. 8505C flank tumors were established by injecting 16106 cells in 100 mL PBS into the subcutaneous flanks of nude mice. When tumors reached 6 mm in mean diameter, mice (n = 7–8 per group) were treated with BEZ235 (50 mg/kg) or placebo by oral gavage daily for 25 days. Tumor dimensions were serially measured with electronic calipers twice a week, and the volumes were calculated by the formula 2 a 6b60.4, where a represents the smallest diameter and b is the perpendicular diameter. The body weight of each animal was followed as a marker of toxicity. Tumor levels of p-AKT, p-S6 ribosomal protein, PCNA and caspase-3 were evaluated in mice treated with a single dose of BEZ235 (50 mg/kg). At indicated periods, carbon dioxide was used for euthanasia, tumors were then harvested, mixed with protein extraction buffer (GE Healthcare), homogenized and sonicated on ice. After centrifugation, clarified supernatants were aliquoted and stored at 280uC until western blot was performed. Tumors were also fixed in 10% formalin and paraffin embedded. Sections (5 mm) were incubated with rabbit p-S6ribosomal protein antibody (1:100) at room temperature for 30 minutes, followed by poly-horseradish peroxidase anti-rabbit IgG reagent, and diami- nobenzidine utilized to visualize the complex. Sections were counterstained with hematoxylin. This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Chang Gung Memorial Hospital and the protocol was approved by the Committee of Laboratory Animal Center at Chang Gung Memorial Hospital, Linkou (Permit Number: 2008112401). Quantitative analysis of drug interactions and statistical analyses Interactions between BEZ235 and the chemotherapeutic drugs for each cell line were determined by calculating the combination index (CI) of Chou-Talalay equation where CI,1 is synergism, = 1 is addictive, and .1 is antagonism [28,29]. The dose-effect analysis was produced using the computer software, CompuSyn [27,30], following the dose and effect data entries. The dose reduction index (DRI) was also determined. DRI represents the Thyroid Cancer and NVP-BEZ235 fold dose-reduction permitted by the combination, for a given effect level, when compared with each drug alone [30]. Comparisons were performed when appropriate using two- sided Student’s t test, and correlations using Pearson’s coefficients (Excel, Microsoft). Results were expressed as the mean 6 SE. In conclusion, BEZ235 effectively inhibits the proliferation of four different histologic types of thyroid cancer. The therapeutic effect and safety profiles are favorable in nude mice bearing 8505C xenograft tumors. Importantly, BEZ235 synergistically enhances the therapeutic effect of paclitaxel in treating ATC. These data support future clinical trials investigating the utility of BEZ235 as an agent to treat patients with refractory thyroid cancer. Supporting Information Appendix S1 (TIF) Basal expression of p27 in thyroid cancer cell lines. Appendix S2 Reported data of genetic alterations in thyroid cancer cell lines and Dm of BEZ235. (TIF) Figure S1 BEZ235 consistently inhibits mTORC1 signaling and activates p-ERK1/2. A, p-AKT (Thr308) was transient reduced by 2 hours in TT and BHP7-13 and increased by 4 hours in 8505C and KAT4C. B, p-AKT (Ser473) was reduced by 2 and 8 hours in TT and BHP7-13 and increased by 2 hours in 8505C and KAT4C. C, p-4E-BP1 (Thr70) was consistently reduced by 2 hours in 3 cell lines. D, p-4E-BP1 (Thr37/46) was reduced by 2 hours. The extent of repression is statistically significant from 2 to 24 hours in BHP7-13 compared with baseline level. E, p-S6 ribosomal protein (Ser235/236) was decreased by 2 hours. The inhibition was significant in 8505C and TT from 4 to 24 hours compared with basal level. F, p-ERK1/2 (Thr202/Tyr204) was activated by 2 to 4 hours in four cell lines. * denoted P,0.02. (TIF) Figure S2 BEZ235 inhibits p-S6 ribosomal protein (Ser235/ 236) and activates p-ERK1/2 and p27 in KAT4C. A, p-AKT (Thr308), p-AKT (Ser473) and p-ERK1/2 (Thr202/Tyr204) was increased by 4 hours and persisted for more than 24 hours. p-S6 ribosomal protein (Ser235/236) was reduced from 8 hours through over 24 hours. B, p27 was increased by 24 hours. p53, p21 and cyclin D1 were increased by 4 to 24 hours. (TIF) Figure S3 BEZ235 activates p27 expression in thyroid cancer cell lines. A, p53was decreased by 2 to 4 hours in 8505C and TT and increased in KAT4C by 8 hours. B, p21 was repressed in TT and BHP7-13 and increased in KAT4C by 8 hour. C, p27 was increased in all cell lines. The elevation of p27 achieved statistical significance at 8 hour in TT. There was no detectable signal in basal 8505C cells, therefore the reference data was at 4 h. D, Cyclin D1 was gradually decreased in 8505C and BHP7-13 and increased at certain time points in KAT4C and TT. There was no detectable signal in untreated KAT4C, therefore the reference data was 8 h. * denoted P = 0.019 (t-test). (TIF) Figure S4 BEZ235 degrades caspase-3 in vitro. Quantification of immunoblot showed caspase-3 was decreased in KAT4C and KAT18 at 72 hours at doses ranged from 6.25 to 100 nmol/L. (TIF) Figure S5 BEZ235 represses the expression of p-AKT, p-S6 ribosomal protein and capase-3 in vivo. The effects of BEZ235 on p-AKT, p-S6 ribosomal protein, PCNA and capase-3 in vivo. Quantification of immunoblot showed BEZ235 greatly repressed PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e46726