Establishment and Authentication of Thyroid Tumor Cell Lines

Cell lines were isolated as previously described [16 (link)]. Hras1, H340T, and H245T tumor cell lines were established from thyroid tumors of Hras^G12V/Pten^−/−/TPO-Cre mice of a pure 129/svJ genetic background. Thyroid tumors and wild-type thyroid glands were dissected and minced, followed by digestion in a solution of 1 mg/mL collagenase Type I (Sigma, St. Louis, MO, USA) and 1 mg/mL dispase (Gibco, Waltham, MA, USA) in Hank’s Balanced Salt Solution at 37 °C with gentle shaking for 1.5 h. Following digestion, samples were centrifuged at 1200 rpm for 3 min and resuspended in Ham’s F12 medium (Corning, Glendale, AZ, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM L-Glutamine (Gibco), and Penicillin/Streptomycin/Fungizone (Sigma). The samples were then plated into tissue culture flasks and maintained at 37° Celsius in 5% CO₂. To ensure removal of contaminating stromal cells and outgrowth of the pure tumor cell lines, all cell lines were passaged at least 5 times after plating and then genotyped using primers specific for Pten and Pten recombination [21 (link)]. Cell lines were authenticated using Short Tandem Repeat (STR) DNA profiling (DDC Medical) according to ANSI guidelines (ASN-0002). Ten mouse STR loci were analyzed for each sample. Loci and STR profiling results are listed in Table S2.