Human epithelial cell medium

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)].

Human pulmonary microvascular endothelial cells (HULEC5a; ATCC-CRL-3244) were purchased from ATCC (Manassas, VA, USA) and cultured in an MCDB131 medium (Calsson Labs, Smithfield, UT, USA) supplemented with 10% FBS (Gibco, Waltham, MA, USA), 1% Penicillin/Streptomycin (Gibco, Waltham, MA, USA), 10 mM L-Glutamine (Gibco, Waltham, MA, USA), 10 ng/mL Epidermal Growth Factor (GenScript, Piscataway, NJ, USA), and 1 µg/mL hydrocortisone (Alfa Aesar, Waltham, MA, USA). Primary human pulmonary artery endothelial cells (HPAEC) were purchased from Lifeline Cell Technology (Carlsbad, CA, USA) and cultured in an endothelial cell medium (ScienCell, Carlsbad, CA, USA), supplemented with 5% fetal bovine serum (FBS), 1% endothelial cell growth supplement, and 1% antibiotic solution (Penicillin/Streptomycin) (ScienCell, Carlsbad, CA, USA). Primary human alveolar epithelial cells (HPAEpiC) were purchased from ScienCell (Carlsbad, CA, USA) and cultured in a human epithelial cell medium (Cell Biologics, Chicago, IL, USA), supplemented with the epithelial cell medium supplement kit (Cell Biologics, Chicago, IL, USA). HULEC5a cells were used at a passage number <20, and HPAEC or HPAEpiC at a passage number <15. Separate passages from a given lot were considered biological replicates.