Supplemental material and methods Reagents. Hydralazine was purchased from Sigma-Aldrich. Cell Culture. The human thyroid follicular carcinoma cell lines FTC-238, FTC-236 and FTC- 133, human thyroid medullary carcinoma cell lines MTC-1.1, and human Hurthle thyroid cancer cell line XTC-1 were cultured as described previously (1). The FTC-238, FTC-236, MTC-1.1 and XTC-1 cell lines were obtained from Dr. Orlo H. Clark (University of California at San Francisco, San Francisco, CA), and the FTC-133 cell line was obtained from Peter Goretzki (Lukaskrankenhaus of the Heinrich-Heine University, Dusseldorf, Germany). Immunohistochemistry analysis of Mig-6. Human normal thyroid and PTC tissue specimens sections were deparaffinized, rehydrated through graded alcohol, and processed using a streptavidinbiotin-peroxidase complex method. Antigen retrieval was done by heating sections using a pressure boiler in 10 mm sodium citrate buffer (ph 6) for 20 minutes. Following quenching of endogenous peroxidase activity and blocking of nonspecific binding, sections were incubated with rabbit anti-human Mig-6 antibody (Sigma-Aldrich) at 4 o C overnight at a 1:100 dilution. The secondary antibody was biotinylated goat anti-rabbit antibody (Vector Laboratories) used at a dilution of 1:600 for 30 minutes at room temperature. After further washing with PBS, sections were incubated with StrepABComplex/horseradish peroxidase (1:100; Dako, Carpinteria, CA) for 30 minutes at room temperature. Immunolocalization was done by exposure to 0.05% 3,3 -diaminobenzidine tetrahydrochloride as the chromogen. Normal serum was used in place of the primary antibody as a negative control. Slides were counterstained with hematoxylin before dehydration and mounting.
Case No. Supplemental Tables Supplemental Table 1. Mig-6 expression and clinicopathologic characteristics from the study sample. Source Mig-6 level Age (> 40 years) Tumor size (> 2 cm) Extrathyr oidal extension LVI Original histological pattern #1 BWH - + - + - Papillary thyroid carcinoma, classical #2 BWH - + + - - Papillary thyroid #3 BWH - - + - - Papillary thyroid #4 BWH - + + - - Papillary thyroid #5 BWH + + - - - Papillary thyroid #6 BWH - + + + + Papillary thyroid #7 BWH - + - - - Papillary thyroid #8 BWH - + + - - Papillary thyroid carcinoma, tall cell #9 BWH - + - - + Papillary thyroid carcinoma, diffuse sclerosing #10 BWH + + - - - Papillary thyroid carcinoma, mixed classical and follicular s, with sclerosing features #11 BWH + + - - - Papillary thyroid carcinoma, tall cell and classical #12 BWH - - - - + Papillary thyroid carcinoma, mixed classical and follicular
#13 BWH - + + - + Papillary thyroid carcinoma, encapsulated solid and follicular s #14 BWH - + - + + Papillary thyroid carcinoma, mixed tall cell and follicular with sclerosis #15 BWH + + - - + Multifocal papillary thyroid carcinoma, follicular #16 BWH + + + - - Papillary thyroid carcinoma, classical #17 BWH - + + - - Papillary thyroid carcinoma, encapsulated mixed columnar and follicular cell s #18 BWH + - + - - Papillary thyroid carcinoma, diffuse sclerosing #19 UCSF - - + + + Papillary thyroid carcinoma, multicentric #20 UCSF + + - - - Papillary thyroid #21 UCSF - - + + + Papillary thyroid carcinoma, classical #22 UCSF - + + + - Papillary thyroid carcinoma, classical #23 UCSF - + + - - Papillary thyroid, multicentric #24 UCSF - - + - - Papillary thyroid adenocarcinoma #25 UCSF - - + - + Papillary thyroid adenocarcinoma #26 UCSF - - + + - Papillary thyroid, multicentric #27 UCSF - - + - - Papillary thyroid carcinoma, classical #28 UCSF - + - + - Papillary thyroid carcinoma, classical #29 UCSF - + - - - Papillary thyroid
, #30 UCSF - + - - - Papillary thyroid carcinoma, multicentric #31 UCSF - - - - - Papillary thyroid carcinoma, classical BWH= Brigham and Women's Hospital; UCSF=University of California San Francisco; LVI = Lymphovascular invasion Supplemental Table 2. Comparison of Mig-6 expression with clinicopathological characteristics. Age < 40 Age > 40 p value Mig-6 (-) 8 16 Mig-6 (+) 1 6 > 0.05 Total PTC specimens: 31 Tumor size < 2 cm Tumor size > 2 cm p value Mig-6 (-) 5 19 Mig-6 (+) 5 2 = 0.01 Total PTC specimens: 31 LVI (-) LVI (+) p value Mig-6 (-) 16 8 Mig-6 (+) 6 1 > 0.05 Total PTC specimens: 31 Extension (-) Extension (+) p value Mig-6 (-) 17 7 Mig-6 (+) 7 0 > 0.05 Total PTC specimens: 31
Supplemental Figure legends Supplemental Fig. 1. Representative examples of Mig-6 immnohistochemical analysis on human normal thyroid and PTC tissue specimens. Expression level of Mig-6 in normal thyroid and PTC tissues resected from patient #3, #4 and #13 were assayed using immunohistochemical analysis. All images were visualized by phase contrast microscopy. (Original magnification, 400). Supplemental Fig. 2. Mig-6 expression in thyroid cancer cell lines. (C) XTC-1, MTC-1.1, 8505-C, TPC-1, FTC238, FTC-236 and FTC-133 cell lysates (150 µg protein) were collected and endogenous Mig-6 expression levels were assayed by Western Blotting. Re-probing actin was done to ensure equal loading. Supplemental Fig. 3. Hydralazine decreases Mig-6 promoter methylation levels and increases Mig-6 protein expression level in Mig-6 promoter hypermethylated thyroid cancer cell lines MTC 1.1, FTC-238, FTC-236, FTC-133 cells. (A) MTC-1.1, FTC238, FTC-236 and FTC-133 cells were treated with or without 40 μm of hydralazine for 72 h. Genomic DNA of each cell line were collected and subject to MSP analysis of Mig-6 promoter. Appropriate primers were used and MSP products were resolved on a 2% agarose gel. (B) MTC-1.1, FTC238, FTC- 236, FTC-133 and 8505-C cells were treated with or without 40 μm of hydralazine for 72 h. Cell lysates were collected and endogenous Mig-6 expression levels were assayed by Western Blotting. Re-probing actin was done to ensure equal loading. Supplemental Fig. 4. NF-κB activation status in normal thyroid tissue specimens. Representative examples of Western Blots analysis of p65 cytoplasmic and nuclear localization in normal thyroid tissue and PTC specimens resected from patients as indicated. Cytosolic (C) and
nuclear (N) extracts (100 µg protein) of specimens were prepared as described in Materials and methods, and subjected to Western blotting analysis with p65 antibody. Re-blotting of OCT-1 and Hsp90 were used as internal controls for nuclear and cytosolic extracts to ensure equal loading. Relative intensity of each band corresponding to 65 kd (normalized to OCT-1 or Hsp90) was analyzed by densitometry analysis using Image J software and shown in the figure. Supplemental references 1. Soh EY, Duh QY, Sobhi SA, Young DM, Epstein HD, Wong MG, Garcia YK, Min YD, Grossman RF, Siperstein AE, Clark OH 1997 Vascular endothelial growth factor expression is higher in differentiated thyroid cancer than in normal or benign thyroid. J Clin Endocrinol Metab 82:3741-3747