Flow cytometry For cell cycle analysis, cells were fixed with 70% ice-cold ethanol and stained with a solution containing 5 mg/ml propidium iodide (Sigma Aldrich, St. Louis, MO, USA) and 100 mg/ml RNase A (Fermentas, St Leon-Rot, Germany). The results were analyzed in FlowJo (Tree Star, OR, USA). Apoptosis assay Cells were fixed with 4% paraformaldehyde at room temper- ature and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate (2 min) on ice. TdT mediated dUTP Nick End Labeling (TUNEL) was performed using the ‘‘In situ cell death detection kit, fluorescein’’ (RocheH), following the manufacturer’s instructions. Each experimental condition was performed three times and the number of positive nuclei in a total of 500 cells was determined in a fluorescence microscope. JAK1 and JAK2 silencing Pre-designed small interference RNAs (siRNAs) targeting human JAK1 (NM_002227) and JAK2 (NM_004972; NM_004972; NM_004972) were purchased from Qiagen, Hilden, Germany. The AllStars negative control with the following target sequence: 59- AATTCTCCGAACGTGTCACGT-39 was also from Qiagen. Cells were transfected with Lipogen (InvivoGen, Toulouse, France) and 25– 50 nM of the siRNA. JAK1 and JAK2 down-regulation was verified after 48 hours. Proliferation assay For analysis of cell proliferation, bromodeoxyuridine (BrdU) incorporation into cellular DNA during S-phase was determined. Cells were incubated with 10 mM bromodeoxyuridine (BrdU) for 1 hour, fixed with 4% paraformaldehyde, treated with HCl 2 M and incubated with a mouse anti-BrdU secondary antibody (DAKO, Glostrup, Denmark), followed by incubation with secondary anti-mouse antibody, conjugated with Alexa 594 (Invitrogen, Carlsbad, USA). Cells with nuclear positivity were counted in a total of 500 cells, under a fluorescence microscope. RET kinase assay The kinase activity of RET was determined using 2 ng/mL of recombinant RET and 1 mg/mL of IGF1 as substrate, in the presence of ATP (50 mM), for one hour, at room temperature (Promega Corp., Madison, WI, USA). After ADP to ATP conversion, ATP was converted into light using the ADP-Glo Kinase Assay (Promega Corp.), according to manufacturer’s instructions. The luminescent signal was recorded in a luminometer. In vivo tumorigenicity assay Each experimental group included 5 female homozygous athymic nu/nu mice (Harlan Laboratories), all littermates. TPC- 1 cells were harvested, mixed with an equal volume of matrigel (BD Biosciences, Franklin Lakes, NJ, USAhttp://en.wikipe- dia.org/wiki/United_States) and injected subcutaneously into the flanks of the mice. When tumors had approximately 3 0.5 cm , mice were treated with the inhibitors. AZD6244 and AZD1480 were dissolved in 0.5% (wt/vol) hydroxyl-propyl- methylcellulose, 0.2% (vol/vol) Tween 80 (Sigma Aldrich, St. Louis, MO, USA). AZD6244 was administered once daily by oral gavage at a dose of 25 mg/kg. AZD1480 was administered by oral gavage bidaily, at 30 mg/kg. Tumor dimensions length (l) and width (w) were measured and tumor volume was calculated by the formula for ellipsoids V(tumor) = plw2/6 [44]. The animals were sacrificed by CO2 asphyxiation, following all the institutional AZD1480 Inhibits Thyroid Tumorigenesis procedures to avoid animal suffering. No signs of toxicity were detected during the drug treatments. Immunohistochemistry Immunohistochemistry was carried out by the streptavidin- biotin-HRP method. Antigen retrieval was performed in citrate buffer (pH 6.0) (Ki67, phospho-ERK1/2) or Tris-EDTA (pH 9.0) (phospho-STAT3, phospho-S6) for 15 minutes, under boiling temperature, in a microwave. After peroxidase blocking in a 3% hydrogen peroxide solution and non-specific binding blocking using the Large Volume Ultra V Block reagent (Thermo Scientific/Lab Vision, Fremont, USA), samples were incubated with the respective primary antibodies (the same used in western- blotting). Tyramide Signal Amplification (TSA) Biotin System (Perkin-Elmer, Foster City, USA) was used for phospho-STAT3 signal amplification as well as for TUNEL (Roche Diagnostics Corp., Basel, Switzerland) staining, according to manufacturer’s instructions. Ki67 RTU was from Novocastra (now Leica Microsystems, Wetzlar, Germany). Meca-32 was custom made by Developmental Studies Hybridoma Bank, IA, USA. As secondary reagents, we used a labeled streptavidin-biotin im- munoperoxidase detection system (Thermo Scientific/Lab Vision, Fremont, USA) followed by DAB (3,39-diaminobenzidine) devel- oping. The results were evaluated under a brightfield microscope and scored semiquantitatively (phospho-STAT3, phospho-S6, phospho- ERK1/2) or quantitatively (Ki67, Meca-32). For semiquantitative analysis, both the intensity (0, 1, 2, 3) and the percentage of positive cells (0–5%:1; 6–25%:2; 26–50%:3; 51–75%:4; 76–100%:5) were considered. The respective scores were multiplied and grouped in four classes: negative (score:0), low (+1; score 1–3), moderate (+2; score 4–8) and high (3+; score 9–15). For meca-32, the number of vessels was counted in at least 5 representative 400x optical fields. For Ki67 and TUNEL, at least 1000 cells were counted and the percentage of positive cells was determined. Statistical analysis Statistical analysis was done in StatView (SAS Institute) and GraphPad, using t-Student test and ANOVA. Results were considered statistically significant when p,0.05 (*, p,0.05; **, p,0.001;***, p,0.0001). Supporting Information Figure S1 Sensitivity of BRAF and RAS- mutated thyroid cancer cell lines to JAK and MEK inhibition. (A) K1 (BRAFV600E) and C643 (HRASG13R) cell lines were treated with AZD6244 (1 mM), AZD1480 (1 mM), and a combination of both drugs (1 mM each) for the indicated time. Growth was determined by the SRB assay. ***p,0.0001. (TIF) Figure S2 Dose-response curves from RET-mutated thyroid cancer cell lines treated with AZD1480. Cell lines were treated with the indicated concentrations of the drug for 48 and 72 hours. Results represent mean 6 SE of three independent experiments. (TIF) Figure S3 AZD1480 inhibits RET kinase activity. Re- combinant RET was incubated with its substrate, IGF1, in the presence of ATP and DMSO (control) or different concentrations of AZD1480 (from 0.001 mM to 2 mM). The data represent percent activity of RET after compound treatment. (TIF) PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e46869