Supporting Information

Transcription

1 Supporting Information Franco et al /pnas SI Materials and Methods Drug Administration. PD was dissolved in 0.5% (wt/vol) hydroxyl-propyl-methylcellulose, 0.2% (vol/vol) Tween 80 (Sigma) and administered to mice once daily by oral gavage at a dose of 25 mg/kg. L-T4 was dissolved in 4 mm NaOH. Mice up to 3 wk of age were treated with L-T4 via daily s.c. injections at a dose of 0.2 mg/kg. After weaning, mice were treated with L-T4 via drinking water, delivering an estimated dose of 0.6 mg kg 1 d 1. TshR-KO mice were supplemented with 0.1 mg kg 1 d 1 of L-T4 via drinking water after weaning. Hypothyroidism was induced in FR-Hras G12V / TPO-Cre mice at 4 6 wk of age by the administration of 0.1% 6- propyl-2-thiouracil (PTU) (Sigma), given in the drinking water. Real-Time Reverse Transcription PCR. Thyroid lobes were surgically removed and immediately placed in liquid N 2. RNA was isolated using TRIzol (Invitrogen) and 0.6 μg was reverse transcribed with SuperScript III (Invitrogen) in the presence of random hexamers to generate cdna. Quantitative reverse transcription PCR (RT- PCR) was done using Power SYBR Green PCR Master Mix (Applied Biosystems) and primer pairs for β-actin (5 -ctgaaccctaaggccaaccgtg-3 and 5 -ggcatacagggacagcacagcc-3 ), thyroglobulin (Tg) (5 -tgtcccaccaagtgtgaaaa-3 and 5 -ccaaggaaagcttgttcagc-3 ), sodium iodide symporter (NIS) (5 -gctcagtctcgctcaaaacc-3 and 5 -cgtgtgacaggccacataac-3 ), thyroid peroxidase (TPO) (5 -tgacttccaggagcacacag-3 and 5 -gcaagttcagtgatgccaga-3 ), pax8 (5 -cagaaggcgtttgtgacaatga-3 and 5 -tgcactttggtccggatgat-3 ), ttf1 (5 - tccagcctatcccatctgaact-3 and 5 -caagcgcatctcacgtctca-3 ), and TshR (5 - ctctcttacccgagccactg-3 and 5 -ttgtcacccggatcttcttc-3 ). The cycle threshold values for β-actin and the target gene were determined with an Ependorf Thermocycler and used to calculate the normalized relative expression, using the QGENE program (1). Histology and Immunohistochemistry. Immunohistochemistry was performed with the following antibodies on paraffin-embedded sections: perk (Cell Signaling; 1 μg/ml), pmek (Cell Signaling; 0.5 μg/ml), and Ki67 (Vector Laboratories; 0.05 μg/ml). Thyroid Imaging. High-resolution MR imaging of the transgenic mouse thyroid tumors was done on a 200-MHz Bruker 4.7T Biospec scanner equipped with a 400 mt/m gradient coil (Bruker Biospin MRI). Mice were anesthetized with 1% isoflurane gas during scanning and monitored using a small animal physiological monitoring system for respiration (SA Instruments). All of the scanning was done by a custom-built 26-mm ID birdcage resonator capable of uniform quadrature RF excitation and acquisition (Stark Contrast MRI Coils Research). T2-weighted scout images along three orthogonal orientations were first acquired for animal positioning. Mouse thyroid tumor images were then acquired using T2-weighted fast spin-echo rapid acquisition with relaxation enhancement (RARE) sequence with a 0.4-mm-thick coronal slice, a mm field of view, and a spatial resolution of mm. We used the following acquisition parameters: time of repetition = 2 s, echo time = 45 ms, RARE factor 8, with an average of 40 scans and 32 min of acquisition time. H&E staining confirmed that the tumors occupied the entire thyroid gland; therefore the volume of the entire thyroid gland was determined pre- and posttreatment using the formula V = d (sum (Ai) 0.5 (A1+An)), where d is the slice thickness and Ai (i = 1,..., n) is the tumor area in each slice. Radioimmunoassays (RIA). Blood from mice was collected immediately after euthanasia with CO 2 and centrifuged at maximum speed at 4 C for 15 min, and serum was removed and stored at 70 C until assayed. Serum TSH levels were determined as previously described (2). The lower limit of detection for TSH in this assay is 10 mu/l. Samples with high TSH levels were diluted with TSH-deficient mouse serum so that all measurements were within the linear part of the standard curve. The serum levels of total thyroxine were measured using an antibody-coated tube RIA (Seamens Medical Solutions Diagnostics) adapted for mouse serum. The lower limit of detection for T4 in this assay is 0.25 μg/dl. Assay for recombination of LSL-Braf targeted allele. This assay has previously been described (3, 4). Briefly, the following primers were used for detecting recombination in mouse tissue: Primer 1: 5 -AGTCAATCATCCACAGAGACCT-3. Primer 2: 5 -GCCCAGGCTCTTTATGAGAA- 3. Primer 3: 5 -GCTTGGCTGGACGTAAACTC-3. Primer 1 + 2: Detects the wild-type allele yielding a product of 466 bp. Primer 1 + 2: Also detects Cre-recombined Braf allele (Lox- Braf V600E ) yielding a product of 518 bp. This product is larger than the wild-type allele due to the presence of the LoxP site that remains after Cre-mediated recombination. Primer 1 + 3: Detects the LSL-Braf V600E allele yielding a product of 140 bp. 1. Francis-Lang H, et al. (1992) Multiple mechanisms of interference between transformation and differentiation in thyroid cells. Mol Cell Biol 12: De Vita G, et al. (2005) Dose-dependent inhibition of thyroid differentiation by RAS oncogenes. Mol Endocrinol 19: Dhomen N, et al. (2009) Oncogenic Braf induces melanocyte senescence and melanoma in mice. Cancer Cell 15: Mercer K, et al. (2005) Expression of endogenous oncogenic V600EB-raf induces proliferation and developmental defects in mice and transformation of primary fibroblasts. Cancer Res 65: of5

2 Fig. S1. TPO-Cre mediated recombination of the LSL-Braf allele in WT and TshR-KO mice. (A) Approach used to express endogenous levels of Braf V600E off the targeted Braf gene locus in mice. The targeting vector contained a cassette with three LoxP sites (black arrows) bracketing a Braf WT minigene (MG) encoding exons and a neomycin selection marker between exons 14 and a mutated exon 15 of the Braf oncogenic allele. Homologous recombination between the targeting vector and the WT allele in embryonic stem cells generated the LSL-Braf V600E allele. Expression of TPO-Cre recombinase allows deletion of the LSL cassette and endogenous expression of the Lox-Braf V600E allele in the thyroid. Locations of PCR primers used for genotyping and to detect recombination are illustrated (arrows 1 3). (B) PCR-mediated genotyping of the Braf alleles in DNA extracted from thyroids of LSL-Braf V600E /TPO-Cre at 3 4 d(n =4)and7 10 d postnatally (n = 4). Each paired sample represents DNA from a single thyroid. Only two of the four samples at days 3 and 4 showed recombination (Upper band), whereas at day 7 all four had detectable recombination, with Lox-Braf/WT-Braf allelic ratios approximating 1:1. (C) PCR-mediated genotyping of the Braf alleles in DNA extracted from thyroids or tails of representative LSL-Braf V600E /TPO-Cre and LSL-Braf V600E /TPO-Cre/TshR-KO mice. (D) IHC images of pmek staining in thyroid sections of LSL-Braf V600E /TPO-Cre and LSL-Braf V600E /TPO-Cre/TshR-KO mice. pmek staining is uniformly present in both LSL-Braf V600E /TPO-Cre and LSL-Braf V600E /TPO-Cre/TshR-KO mice, suggesting Braf recombination is ubiquitous and present in most thyrocytes. 2of5

3 Fig. S2. LSL-Braf V600E /TPO-Cre mice are hypothyroid and have reduced expression of thyroid-specific genes. (A) Weight of LSL-Braf V600E /TPO-Cre and WT littermates at 5 wk. (B) TSH and free T4 levels in 5-wk-old LSL-Braf V600E /TPO-Cre and control littermates. Bars represent mean ± SEM; P < vs. wild type. (C) Quantitative RT-PCR analysis of mrna levels of Tg, Tpo, Nis, TshR, Pax8, and Ttf1 in thyroid glands of 5-wk-old LSL-Braf V600E /TPO-Cre and age-matched wildtype littermates. Bars represent mean relative expression ± SEM of the indicated genes after normalization to β-actin. *P < 0.01 vs. wild type. 3of5

4 Fig. S3. TSH suppressive therapy with high-dose L-T4 does not prevent Braf-induced PTC. (A) H&E staining of sections of wild-type and LSL-Braf V600E /TPO-Cre thyroid lobes from 3-d-old mice. (Magnification, 40.) (B) Serum TSH levels in WT (n = 15) and LSL-Braf V600E /TPO-Cre mice (n = 6) at 3 d (mean ± SEM; P = not significant, NS). (C) Quantitative RT-PCR analysis of mrna levels of Nis, Tpo, and TshR in thyroid glands of 3-d-old LSL-Braf V600E /TPO-Cre and age-matched WT littermates. Bars represent mean relative expression ± SEM after normalization to β-actin. (D) Ki67 index of thyroid tissues at postnatal days 0 and 7 in the indicated genotypes. (E) TSH and free T4 levels in 3-wk-old WT, LSL-Braf V600E /TPO-Cre, and LSL-Braf V600E /TPO-Cre mice treated with L-T4 from day 3 postnatally. Bars represent mean ± SEM. Serum TSH levels in LSL-Braf V600E /TPO-Cre treated with L-T4 were below the level of detection of the assay and are reported as 5 miu/l, which is 50% of the detection limit of the assay. P < vs. wild type. (F) Suppression of TSH beginning at postnatal day 3 does not inhibit PTC development. Thyroid sections are shown from LSL-Braf V600E /TPO-Cre mice treated with vehicle or L-T4 for 3 wk. (Magnification, 40.) No difference in size or tumor histology was observed. 4of5

5 Fig. S4. Effects of thyroid-specific endogenous expression of HrasG12V on thyroid function. (A) TSH and T4 levels in 5-wk-old WT (n = 16) and FR-Hras G12V / TPO-Cre littermates (n = 16). (B) Quantitative RT-PCR analysis of mrna levels of Tg, Tpo, Nis, TshR, Pax8, and Ttf1 in thyroid glands of 5-wk-old FR-Hras G12V / TPO-Cre and age-matched WT littermates. Bars represent average relative expression, after normalization to β-actin, of the indicated genes. No significant differences in expression were detected between any groups. Fig. S5. Treatment with PTU does not induce thyroid tumors in FR-Hras G12V /TPO-Cre mice. WT, FR-Hras G12het /TPO-Cre, and FR-Hras G12Vhom /TPO-Cre mice were treated with vehicle or PTU for 6 or 20 wk. (A) Representative H&E images of thyroid lobe sections from vehicle and PTU-treated mice. Treatment with PTU led to the formation of benign goiters in all genotypes, but no tumors developed. (Magnification, 40.) (B) Representative IHC images of Ki67 staining. PTU treatment led to a significant increase in cellular proliferation in all groups, without any significant difference between them. 5of5