Rpmi 1640 w glutamax 1

All plasmids (listed in Supplementary Table S1) were constructed using the Gibson assembly method. The human embryonic kidney cell line, HEK293T (ATCC CRL-3216), was cultured in complete media (DMEM; 1 g/L glucose, 2 mM L-glutamine, 10% heat-inactivated FBS (fetal bovine serum; Gibco)) with 5% CO₂ at 37 °C. We used plasmid pcDNA3 (Invitrogen) to express INSRTR proteins. Renilla luciferase (phRLTK, Promega) was used as a transfection control in the dual luciferase assay. Jurkat, Raji, and SK-BR-3 cell line were cultured in complete media (RPMI 1640 W/GLUTAMAX-I, Gibco, 10% heat-inactivated FBS (Gibco)) with 5% CO2 at 37 °C.

Two human papillary thyroid cancer cell lines (TPC1 and BCPAP) were used. The TPC1 cell line was a gift from Dr Velázquez Henar (Autonomous University of Barcelona, Spain) and BCPAP cell line was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen, (German) (DSMZ Cat# ACC-273 (established from a metastasizing papillary thyroid carcinoma)). TPC1 and BCAP were cultured in RPMI 1640 w/Glutamax I (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and Dulbecco’s modified Eagle’s medium (DMEM) culture medium (Gibco, Thermo Fisher Scientific), containing 10% fetal bovine serum (Gibco, Thermo Fisher Scientific), 1% L-glutamine 200 mM (Gibco, Thermo Fisher Scientific), 1% Penicillin-Streptomycin 10,000 U/mL (Gibco, Thermo Fisher Scientific) and 1% Amphotericin B 250 μg/mL (Gibco, Thermo Fisher Scientific) in a 37 °C incubator with 5% CO₂.