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IJC_25275_sm_suppinfofig1.tif16348KSupplemental Figure 1: Efficiency of hNAA10 knockdown in thyroid cell lines. Different thyroid cells were seeded in 6-well plate and transfected with sihNAA10-1 and mRNA level of hNAA10 was analysed by qRT-PCR 48 h later. Values are mean ± SD of independent experiments, presented in % of hNAA10 normalized level (to a reference gene RPLP2) in knockdown cells versus control (cells transfected with non-silencing siRNA).
IJC_25275_sm_suppinfofig2.tif17577KSupplemental Figure 2: Diversity of thyroid cell lines. Relative hNAA10 and hNAA15 mRNA expression (a) was analysed by qRT-PCR. Values are mean ± SD of three replicates, expressed as a number of copies per 1000000 copies of RPLP2. Expression of hNaa10p, hNaa15p and p53 was analysed by Western blotting (b). 50 μg of total protein was loaded per lane; β-tubulin and actin were used as loading controls. Cells were also checked for basic expression level of antiapoptotic proteins Bcl-2 and Bcl-xL.
IJC_25275_sm_suppinfofig3.tif8720KSupplemental Figure 3: Decrease in acetylated β-catenin is not required for cyclin D1 downregulation in hNatA-depleted cells. hNatA knockdown was mediated by sihNAA10 transfections of human thyroid anaplastic carcinoma CAL-62. 72 h later cells were harvested, lysed in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% w/v SDS, 0.5% sodium deoxycholate, 0.1 M Na3VO4, 10 mM NaF, 15 μM TSA and Complete Protease Inhibitor Cocktail mini (Roche Applied Science)]. Protein concentration was determined using microBCA kit, and equal amounts of total protein (500 μg) were incubated with 1μg anti-β-catenin antibodies to immunoprecipitate endogenous β-catenin at 4°C overnight. The immunocomplexes were collected with protein A/G PLUS – agarose (Santa Cruz) and washed 5 times with lysis buffer. The acetylation status of precipitated β-catenin was checked by acetyl-lysine antibodies. IP-complexes were also analysed for association with hNaa10p. β-tubulin was used as a loading control for cell lysates before immunoprecipitation; heavy chains of β-catenin antibodies and β-catenin were loading controls for analysis of IP-complexes. Representative results from several experiments are shown. Similar data were also obtained for human thyroid anaplastic carcinoma 8305C.
IJC_25275_sm_suppinfotables.doc51KSupplemental Table I: TP53 mutations in thyroid cell lines and predicted TP53 function. Supplemental Table II: Sequences of primers used in qRT-PCR.

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