Ftc 133

Human tumor cell lines were cultured in DMEM/10% FCS under a humidified 5% CO₂ atmosphere at 37 °C. The following cell lines were used: A-172, A549, A64-CLS, Hela, HL-60, LNCaP, MCF-7, MG-63, PC-3, SaOs, and SW-480 from Cell Lines Service (Eppelheim, Germany); B-CPAP, BHY, CAL-62, COLO320HSR, H1184, H146, HT29, LoVo, SKN-MC, SW-403, and THP-1 from DSMZ (Braunschweig, Germany); FTC-133, U251-MG, and U373-MG from Sigma-Aldrich (Taufkirchen, Germany); and SW948 and U87-MG from American Type Culture Collection (Manassas, VA, USA), as previously described [18 (link)].

Human follicular thyroid carcinoma cell lines FTC-133 (derived from a lymph node metastasis) and FTC-238 (derived from a lung metastasis) (product numbers 94060901 and 94060902 respectively, Sigma) were included in this study. The mutational landscape of both cell lines was recently published (Landa et al. 2019 (link)), and we confirmed the presence of a homozygous TERT promoter mutation c.228C>T (-124C>T) by Sanger sequencing in both cell lines (data not shown). The cell lines are identical to the source provided by the depositor based on an STR-PCR analysis. Cells were maintained in DMEM supplemented with 5% foetal bovine serum (FBS) in culture dishes. Experiments were conducted after 12–15 passages, allowing a maximum of 15 passages.

We studied two PTC cell lines (BHP7-13 and K1) and two FTC cell lines (FTC-133 and FTC-238). The BHP7-13 cells have been previously described [33 (link)]. The K1, FTC-133, and FTC-238 cells were purchased from Sigma (now Merck, Darmstadt, Germany). All four cell lines were authenticated using short tandem repeat DNA profiling [47 (link),48 ]. The BHP7-13 cells were maintained in Roswell Park Memorial Institute 1640 media supplemented with sodium bicarbonate (2.0 g/L). The K1 cells were maintained in Dulbecco’s Modified Eagle Medium, Ham’s F12, and MCDB 105 (2:1:1) with glutamine (2.0 mmol/L). The FTC-133 and FTC-238 cells were maintained in Dulbecco’s Modified Eagle Medium and Ham’s F12 (1:1) with glutamine (2.0 mmol/L). All media were supplemented with 10% fetal bovine serum, 100,000 units/L of penicillin, and 100 mg/L of streptomycin. All cells were maintained in standard tissue culture conditions (5% carbon dioxide humidified incubator at 37 °C).

The Human follicular thyroid carcinoma (FTC-133) and normal thyroid follicular epithelial cell line Nthy-ori3-1 (Non-tumorigenic) were obtained from Sigma Aldrich. Nthy and FTC-133 cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). At 60–70% confluence, medium was changed to hormone, growth factor and phenol red free DMEM supplemented with 5% charcoal stripped FBS (CTS) before experiments were started. Inhibitors used were added 30 minutes prior to starting the experiment. 17β–estradiol (E2), ICI-182780 (ICI), Y-27632, wortmannin and PD-98059 were from Sigma-Aldrich. 4′,4″,4″′-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol 10 nM (PPT), 2,3-bis-(4–hydroxyphenyl)-propionitrile 10 nM (DPN) were purchased from Tocris Bioscience.

The human follicular thyroid carcinoma cell lines FTC-133 (passages 17–25) and WRO (passages 10–16) and the noncancerous follicular epithelial cell line Nthy-ori 3-1 (passages 10–15) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The human follicular thyroid carcinoma cell line ML-1 (passages 5–9) isolated from recurrence [32 (link)] was used from a laboratory stock. Cells were cultured in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, St. Louis, MO, USA), and 1% penicillin/streptomycin (Life Technologies) at 37 °C and 5% CO₂ until used for experiments. Twenty-four hours before each experiment, a cell density of 1 × 10⁶ cells per flask were seeded in T25 cell culture flasks (Sarstedt, Nümbrecht, Germany) to allow cells to adhere. T25 flasks equipped with glass coverslips and a reduced cell density of 0.5 × 10⁶ cells were used for immunofluorescence staining.
Breast epithelial cells (MCF-10A; ATCC, Manassas, VA, USA), mammary carcinoma cells (MCF-7; ATCC), and prostate carcinoma cells (PC-3, LnCAP; ATCC) for supplemental experiments were cultured accordingly. The MCF-10A cell line was cultured in DMEM/F12 medium supplemented with 0.5% Mammary Epithelial Growth Supplement (MEGS) (Life Technologies).

Human FTC cell line TT2609-C02 was purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ no.: ACC 510). The cell line originated from local tumour recurrence of a secondary RAI refractory FTC. Under culture conditions the cell line demonstrates no relevant ¹²⁵I uptake^{45 (link)}.
The human FTC cell line FTC133 was purchased from Sigma-Aldrich (catalogue no.: 94060901). The cell line was obtained from a patient with metastatic FTC and does not demonstrate iodine uptake under culture conditions^{46 (link), 47} . Authenticity of both cell lines was confirmed by Short Tandem Repeat (STR) DNA profiling.
For lentiviral transduction 2.5 × 10⁶ HEK293FT cells were incubated in DMEM/10% FCS over night at 37 °C and 5% CO₂. Transfection was performed using Lipofectamine2000^TM with envelope plasmid pCMV-VSVG, packaging plasmid psPAX2 (both Addgene, Cambridge, MA, USA) and respective targeting vectors for XIAP (clone V2LHS-94578; mature antisense sequence: TTACAAGTGACTAGATGTC), survivin (clone V2LHS_262484; mature antisense sequence: TTCCTAAGACATTGCTAAG) or GIPZ non-silencing Lentiviral shRNA (all Open Biosystems, Dharmacon, Lafayette, CO, USA).
Lentiviral supernatant was harvested and FTC cell lines transduced using polybrene. Knockdown was validated by western blot analysis.

Human FTC cell lines FTC133 (#94060901, purchased in 2011 from Sigma-Aldrich, Saint Louis, MO) with wild-type PTEN expression (FTC133-PTEN wild type) and FTC236 (FTC236-PTEN null) (#6030202, purchased in 2011 from Sigma-Aldrich, Saint Louis, MO) cells were grown in 50% Ham's F12, 50% DMEM supplemented with 10% fetal bovine serum, 1% glutamine, 0.25% insulin, 0.01U/mL TSH at 37°C with 5% CO₂. The 8505C (#ACC219, purchased in 2011 from DSMZ, Braunschweig, Germany) human papillary thyroid cancer cell lines was grown in EMEM plus 1% non-essential amino acids and 10% fetal bovine serum at 37°C with 5% CO₂. Immortalized lymphoblastoid cell lines (LCLs) obtained from CS patients or normal controls were grown in RPMI1640 supplemented with 20% fetal bovine serum and incubated at 37°C with 5% CO₂.