Gly his lysacetate

Cancer tissues obtained from mPTC^GFP were minced in lysis buffer (Collagenase I (Sigma) 1 mg/ml; Dispase II (Invitrogen) 0.5 mg/ml in PBS) and shaked on an orbital shaker at 37 °C for 60 min. Dissociated tumor cells were filtered with 40 μm cell strainer (#352340, BD Falcon) and centrifugated at 500 g for 5 min, the pellet was resuspended in cold Ham’s F-12 Nutrient Mix (#11765062, Gibco). The isolation of tumor cells was next performed on FACSAria Cell Sorter (Becton Dickinson) according to GFP signal, and positive cells were washed with PBS and then cultured in F-12 medium supplemented with 10% FBS, 1% penicillin/streptomycin, 5 mg/L transferrin (Sigma-Aldrich), 10 mg/L bovine insulin (Solarbio), 3.5 mg/L hydrocortisone (Sigma-Aldrich), 10 mg/L somatostatin (Sigma-Aldrich), 0.02 mg/L Gly-His-Lysacetate (Sigma-Aldrich) and 1 IU/L bovine thyroid stimulating hormone (TSH) (Sigma-Aldrich) related to the 6H medium at 37 °C⁵⁹ . Next, Primary cells treated with 10 μM PLX4032 or 2 μM SCH772984 were subjected to western blot and RT-qPCR analysis. Primary cells from mPTC mice were directly cultured in complete F-12 medium. Thyrocytes were confirmed by immunofluorescence staining of thyroglobulin and representative photos were taken by a fluorescence microscopy.

Cancer tissues obtained from TPO-cre; Braf^V600E; Rosa26-mTmG were minced in lysis buffer (Collagenase I (Sigma), 1 mg/mL; Dispase II (Invitrogen) 0.5 mg/mL in PBS) and shaked on the orbital shaker at 37 °C for 60 min. Dissociated tumor cells were filtered with 40 μm cell strainer (#352340, BD Falcon) and centrifuged at 500 g for 5 min, then the pellet was resuspended in cold Ham’s F-12 Nutrient Mix (#11765062, Gibco). The isolated tumor cells were washed with PBS and then cultured in F-12 medium supplemented with 10% FBS, 1% penicillin/streptomycin, 5 mg/L transferrin (Sigma-Aldrich), 10 mg/L bovine insulin (Solarbio), 3.5 mg/L hydrocortisone (Sigma-Aldrich), 10 mg/L somatostatin (Sigma-Aldrich), 0.02 mg/L Gly-His-Lysacetate (Sigma-Aldrich) and 1 IU/L bovine thyroid stimulating hormone (TSH) (Sigma-Aldrich) related to the 6H medium at 37 °C. Next, Primary cells treated with 10 μM BRAFi (PLX4032), 10 μM ERKi (SCH772984) or AP-1i (T-5224) were subjected to western blot and RT-qPCR analysis.