A total of 81 thyroid tissue specimens obtained from patients undergoing thyroid surgery for thyroid disease at Hospital São Paulo, Federal University of São Paulo, Brazil, were used for this study. Samples were frozen immediately after surgical biopsy and stored at -80°C. The samples included 7 normal thyroid tissues, 21 follicular thyroid adenomas, 14 Hürthle cell adenomas, 15 follicular thyroid carcinomas, 6 Hürthle cell carcinomas and 18 papillary thyroid carcinomas. All tissue samples were obtained with informed consent according to established Human Studies Protocols at Federal University of São Paulo. The study of patient materials was conducted according to the principles expressed in the Declaration of Helsinki.
RNA extraction, cDNA synthesis and quantitative PCR (qPCR)
To investigate the level of ABI3 expression in thyroid tumors, total RNA and cDNA synthesis was performed as previously described . An aliquot of cDNA was used in 20 μl PCR reactions containing TaqMan universal PCR master mix, 10 μM of each specific primer and FAM-labeled probes for the target gene (ABI3) and VIC-labeled probe as the reference gene (S8) (TaqMan®Gene Assays on Demand; Applied Biosystems, Foster City, CA). Gene expression was normalized to the average of S8 expression and relative expression was calculated as described earlier [11, 12].
Correlation of ABI3 and ABI3BPexpression in thyroid tumors
The level of ABI3 expression was correlated with the level of ABI3BP, which was previously investigated in this set of samples .
A follicular thyroid carcinoma cell line (WRO) and a colon cancer-derived HT-29 cell line (ARO)  were grown in DMEM (Invitrogen Corp., Carlsbad, CA) supplemented with 10% FBS (Invitrogen Corp.), 100 units/mL of penicillin and 100 μg/mL streptomycin in a humidified incubator containing 5% CO2 at 37°C [14, 15].
Generation of stable tranfected clones Expressing of ABI3
Plasmid encoding the full-length cDNA of human ABI3 was kindly donated by Dr. Satoru Matsuda (Nagoya University School of Medicine, Nagoya, Japan). To establish cell lines expressing ABI3, 10 μg of DNA construct were transfected into WRO and ARO cells by electroporation using a Gene Pulser II (Bio-Rad Laboratories Inc., Hercules, CA). ARO and WRO cells transfected with pcDNA3.1 vector were used as the negative controls. Clones were isolated after 3 weeks of selection with G418 (800 μg/mL). At least six G418-resistant clones from each transfection were isolated, expanded, maintained on G418 (400 μg/mL) and tested for ABI3 expression by qPCR. To this end, total RNA extracted from each clone was used for cDNA synthesis as described . An aliquot of cDNA was used in a 20 μL PCR reaction containing SYBR Green PCR Master Mix (Applied Biosystems) and 200 nM of each primer for target or reference genes. qPCR was performed in triplicates and the threshold cycle (Ct) was averaged (SD ≤1). Primer sequences for ABI3 and S8 (internal control) were as follows: ABI3 sense 5'-CAGGTGGAAGCCCGTGTAAG-3' and antisense 5`-AGTGGCTAAGGTGCCGATCTC-3', yielding a product of 89 bp; S8 sense 5'-TGAAAGGAAAAAGAATGCCAAAA-3' and antisense 5'-CACTGTCCCGGCCTTGAA-3', yielding a product of 96 bp. Gene expression was normalized to the average of S8 and relative expression was calculated as described [11, 12]. For each cell line, two independently isolated clones that expressed ABI3 at similar levels and two pcDNA3.1 clones were used for further in vitro and in vivo experiments.
About 5 × 106 WRO cells were transfected with 10 μg of the ABI3 DNA construct as described above. Control plates were transfected with pcDNA3.1. After 3 weeks of selection with G418 (800 μg/mL), cells were fixed in 10% acetic acid and 10% of methanol and stained with 1% crystal violet. G418-selected colonies were counted. Each experiment was performed in triplicate.
Stably transfected clones for ARO and WRO were analyzed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay as described [8, 16]. In brief, 2 × 104 cells were seeded in 35-mm plates on day 0. Cell growth was measured from day 1 to 5 by adding 0.5 mg/mL of MTT (Sigma-Aldrich, St. Louis, MO) to the medium at 37°C for 3 hours. The medium was removed and purple formazan crystals were dissolved by adding acid isopropanol. The absorbance of the supernatant was measured at 560 nm.
Quantification of apoptotic cells by annexin-V labeling
To test whether ectopic expression of ABI3 induces apoptosis, 2 × 104 cells were seeded in 35-mm plates and double-stained with Annexin V and Nexin 7-AAD according to the manufacturer's recommendations (Guava Nexin method; Guava Technologies). Cell-associated fluorescence was analyzed by the Guava PCA flow cytometer (Guava Technologies). Results are expressed as the percentage of apoptotic positive cells. Both early apoptotic (annexin V-positive) and late apoptotic (annexin V- and 7 AAD-positive) cells were included in the analysis. Experiments were performed in quintuplicates.
Cell viability assay
ARO Cells (2 × 104) were seeded in 35-mm plates. Cells were mixed with Guava ViaCount Reagent and allowed to stain for 10 minutes (Guava Technologies, Hayward, CA). Viable cells were quantified using a Guava Personal Analyzer (PCA) flow cytometer (Guava Technologies) following the manufacturer's specifications. Experiments were performed in quintuplicates.
Cell cycle analysis
ARO cells (2 × 105) were seeded in 35-mm dishes. After synchronization of the cells by serum starvation for 24 hours, cells were replaced with DMEM medium supplemented with 10% FBS for 24 hours. Cells were fixed in 70% ethanol for 1 hour, labeled with Guava Cell Cycle Assay reagent and analyzed using Guava PCA flow cytometer (Guava Technologies), according to manufacturer's recommendations. Experiments were performed in quintuplicates.
Expression of p21WAF1and E2F1 by qPCR
The transcript levels of p21
WAF1and E2F1 were tested in stably expressing ABI3 ARO and WRO cells and controls, as described .
Western blot analysis
Western blot analysis was performed as described . Briefly, membranes were blocked and incubated overnight at 4°C with anti-phospho-ERK (pERK; dilution 1:1000), anti-phospho-AKT (pAKT; dilution 1:400) and anti-α- Tubulin (dilution 1:1000). Detection was carried out using the SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA).
Senescence-associated (SA) β-gal staining was performed as described . Briefly, ARO and WRO cells (2 × 104) were seeded in 35-mm plates. Cells were washed twice with PBS, fixed for 15 minutes and stained with 1 mg/mL 5-bromo-4-chloro-3-inolyl-b-D-galactoside (X-gal) in buffer (dimethyformamide, 40 mM citric acid/sodium phosphate pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl and 2 mM MgCl2). Cells were incubated at 37°C in 5% CO2 for 18 hours and washed twice with PBS. Cells were examined using a light microscope and counted in 5 optical fields (100×). Data represents mean of an experiment performed in quintuplicates.
Matrigel invasion assay
Cell invasion was analyzed using BioCoat Matrigel Invasion Chamber according to the manufacturer's recommendation (Becton Dickinson, Bedford, MA). WRO cell clones were added to the invasion or control chambers at a density of 2.5 × 104 and, after 24 hours, cells remaining above the insert membrane were removed by gentle scraping with a sterile cotton swab. FBS was used as chemoattractant. Cells that had invaded through the Matrigel to the bottom of the insert were fixed and stained with rapid panoptic LB (Laborclin, Brazil) and mounted. Cells were examined using a light microscope and counted in 3 optical fields (100×). Experimental and control groups were performed in triplicates. The percentage of invasion cells was determined by the mean of cells invading through Matrigel insert membrane divided by the mean of cells migrating through control insert membrane X100.
Cell Migration from spheroids
Because high migration capacity might be correlated with cell spreading and metastasis in vivo, migration from spheroids was assayed as previously described . Briefly, spheroids were prepared by seeding WRO cells in DMEM supplemented with 10% FBS, onto 35-mm tissue culture dishes coated with 0.75% Noble agar. Cells were cultured until spheroids were formed and single spheroids were placed at the center of each well of a 24-well plate. At least 12 single spheroids from each selected clone were cultured. The area covered by cells spreading out from the spheroid was measured every 24 hours for a period of 6 days. The areas of spheroids were calculated as described .
Anchorage-independent growth was assessed by a double-layer soft agar assay. Initially, 60-mm dishes were layered with 0.5% agar and 1× complete medium. Next, ARO cells (1.5 × 104) were suspended in 1× complete medium and 0.35% agar and seeded in triplicate over a bottom layer of solidified agar. The dishes were incubated at 37°C in 5% CO2. After 3 weeks, colonies greater than 20 μm in diameter were counted. Colony formation rate was calculated as percentage of total seeded cells. Two independent experiments were performed.
Nude mouse xenograft model
Four to five week old male athymic nude (nu/nu) mice were maintained according to the guidelines of the Division of Animal Resources at the Federal University of São Paulo. ARO stable cell clones were suspended in sterile PBS to 2 × 106/200 μL and injected subcutaneously into the flank of mice. Mice were then monitored biweekly during three weeks. Tumor volume was calculated by the rotational ellipsoid formula: V = A × B2/2 (A = axial diameter; B = rotational diameter). Tumor tissues were collected and embedded in paraffin for conventional histology or were stored at -80°C.
The relative expression values were log transformed before the application of statistical analysis. Pearson correlation coefficient was used to verify the correlation between ABI3 and ABI3BP expression. In vitro results were log transformed and analyzed by a Student's t test. In vivo results were analyzed by the Wilcoxon test. Significance is presented as p value of <0.05 (*), < 0.01 (**) and < 0.001 (***).