Human thyroid cancer cell lines BcPAP, TPC-1, 8505c, CAL62, SW1736, FRO, BHT101, HTH7 and OCUT1 were obtained and maintained as previously described [20 (link)]. The normal thyroid cells H-6040, isolated from normal human thyroid tissue and cultured in Human Epithelial Cell Medium with the addition of Insulin-Transferrin-Selenium, EGF, Hydrocortisone, L-Glutamine, antibiotic-antimycotic solution, Epithelial Cell supplement, and FBS were purchased from Cell Biologics (Chicago, IL, USA). H-6040 cells were used at passages between 3 and 6.
Transient transfections of TC cells were performed using polyethylenimine according to manufacturer’s instructions (Merck, Darmstadt, Germany). Cells were harvested 48 h after transfection. Electroporation was used (Neon® Transfection System for Electroporation, Life Technologies, Carlsbad, CA, USA) to obtain stably transfected cells [21 (link)].
For RNA interference, we used SMART pools of siRNA from Dharmacon (Lafayette, CO, USA) targeting PD-1 or SHP2. Transfection was carried out by using 100 nM of SMARTpool and 6 μl of DharmaFECT (Dharmacon) for 48 h [22 (link)].
Transient transfections of TC cells were performed using polyethylenimine according to manufacturer’s instructions (Merck, Darmstadt, Germany). Cells were harvested 48 h after transfection. Electroporation was used (Neon® Transfection System for Electroporation, Life Technologies, Carlsbad, CA, USA) to obtain stably transfected cells [21 (link)].
For RNA interference, we used SMART pools of siRNA from Dharmacon (Lafayette, CO, USA) targeting PD-1 or SHP2. Transfection was carried out by using 100 nM of SMARTpool and 6 μl of DharmaFECT (Dharmacon) for 48 h [22 (link)].