Fig 2: Downregulation of TNRC6C-AS1 or overexpression of TNRC6C promotes iodine uptake of TPC1 cells. (A) Downregulation of TNRC6C-AS1 by transfecting of siRNA-TNRC6C-AS1 for 48 h caused significantly increased mRNA expression of NIS, TPO, TSHR and Pendrin in TPC1 cells compared to control cells. (B) Downregulation of TNRC6C-AS1 by transfecting of siRNA-TNRC6C-AS1 for 48 h showed significant increase of protein levels of NIS, TPO, TSHR and Pendrin in TPC1 cells (GAPDH was used as the internal control). (C) Iodine uptake assay showed that iodide accumulation in TPC1 cells was significantly increased after TNRC6C-AS1 knockdown compared to control cells at 6, 12, 24, and 48 h. (D) Overexpression of TNRC6C by transfecting of TNRC6C overexpression plasmid for 48 h caused significantly increased mRNA expression of NIS, TPO, TSHR and Pendrin in TPC1 cells compared to control cells. (E) Overexpression of TNRC6C by transfecting of TNRC6C overexpression plasmid for 48 h showed significant increase of protein levels of NIS, TPO, TSHR, and Pendrin in TPC1 cells compared to control cells. (GAPDH was used as the internal control). (F) Iodine uptake assay showed that iodine accumulation in TPC1 cells was significantly increased after TNRC6C overexpression compared to control cells at 6, 12, 24, and 48 h. vec, empty vector; oe, overexpression vector; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: cAMP response to TSH and 125I-binding activities of HEK 293 cells expressing wild-type TSHR and/or mutated TSHR.(a) Cyclic AMP levels in HEK 293 cells expressing TSHR(W) or TSHR(M) following TSH stimulation. TSH was added to culture medium of HEK 293 cells transfected with 0.5 µg of pcDNA3-TSHR(W) (?-?) or the same amount of pcDNA-TSHR(M) (•-•). After 30 min, cellular cAMP levels were assayed. Data are means ± S.E. of triplicate assays. (b) Cyclic AMP levels in HEK 293 cells expressing TSHR(W) and TSHR(M) following TSH stimulation. TSH was added to culture medium of HEK 293 cells transfected with 0.5 µg of pcDNA3-TSHR(W)+the same amount of pcDNA (?-?), 0.5 µg of pcDNA3-TSHR(W)+the same amount of pcDNA-TSHR(M) (?-?) or 0.5 µg of pcDNA-TSHR(M)+the same amount of pcDNA (•-•). After 30 min, cellular cAMP levels were assayed. Data are means ± S.E. of triplicate assays. (c) 125I-binding activities of HEK 293 cells expressing wild-type TSHR and/or mutated TSHR. HEK 293 cells were transfected with 0.5 µg of pcDNA3-TSHR(W)+same amount of pcDNA (?-?), 0.5 µg of pcDNA3-TSHR(W)+the same amount of pcDNA-TSHR(M) (?-?) or 0.5 µg of pcDNA-TSHR(M)+the same amount of pcDNA (•-•). After 72 h of culture, TSH binding activities were assayed using 125I-bovine TSH. Data are means ± S.E. of triplicate assays.

Fig 4: Interaction between TSHR(W) and TSHR(M).(a, b) Localization of TSHR(W)-eCFP and TSHR(M)-eYFP in HEK 293 cells. HEK293 cells were transfected with 0.5 µg of pcDNA3-TSHR(W)-eCFP (a) or 0.5 µg of pcDNA3-TSHR(M)-eYFP, and after 48 h, fluorescence were observed with an Olympus IX71 fluorescence microscope (×200). Bars indicate 50 µm. (c) cAMP responses of tagged (TSHR(W)-eCFP) and untagged TSHR (TSHR(W)). TSH was added to culture medium of HEK 293 cells transfected with 0.5 µg of pcDNA3-TSHR(W) (?-?) or the same amount of pcDNA-TSHR(W)-eCFP (?-?). 0.5 µg of pcDNA3 was also transfected into HEK293 cells (•-•). After 30 min, cellular cAMP levels were assayed. Data are means ± S.E. of triplicate assays. (d) FRET image of TSHR(W)-eCFP cells fused to TSHR(W)-eYFP. HEK 293 cells were transfected with 0.5 µg of pcDNA-TSHR(W) and the same amount of pcDNA-TSHR-eYFP, and were grown in medium for 72 h for live cell imaging. Left panels show fluorescence of donor, eCFP (upper panel), acceptor, eYFP (lower panel) and FRET image (middle panel) in the absence of TSH. Right panels show donor (upper panel), acceptor (lower panel) and FRET image (middle panel) in the presence of 1 mU/ml TSH. (e) Acceptor/Donor ratio in FRET assay from HEK 293 cells expressing TSHR(W or M)-eCFP and TSHR(W or M)-eYFP in the presence or absence of TSH. Acceptor/donor ratio was measured by Metamorph software in RoIs from 10 different cells expressing TSHR(W)-eCFP and TSHR(W)-eYFP, TSHR(W)-eCFP and TSHR(M)-eYFP, and TSHR(M)-eCFP and TSHR(M)-eYFP in the presence or absence of 1 mU/ml TSH. Bars indicate means ± S.E. *: p<0.01. (f) Cross-linking between TSHR(W) and TSHR(M) in the presence or absence of TSH. HEK 293 cells expressing TSHR(W)-eCFP and TSHR(W)-eYFP, and TSHR(W)-eCFP and TSHR(M)-eYFP were cultured in the presence or absence of TSH for 24 h, and dimethyl suberimidate was added to the medium at a final concentration of 2 mg/ml, followed by further incubation for 30 min. Proteins were electrophoresed, transferred onto nitrocellulose filter, and TSHR was detected with anti-TSHR antibody. BRL: BRL-3A, rat liver cells.

Fig 5: Promotion of TSHR palmitoylation by palmitic acid, leading to the enhancement of the proapoptotic effects of TSH. (a) Sequencing results of the gene missing a base. Deletion of base A caused frameshift mutations and premature termination of translation that could not be translated into the TSHR protein. (b) WB results corresponding to cultured monoclonal cell lines. Each band represents a monoclonal cell line. The red arrows are indicative of stable TSHR knockout cell lines. (c) Expression levels of Cleaved-caspase 9, Cleaved-caspase 3, Bcl2, and Bax determined via western blotting (n = 3) and (d) analyzed. Cleaved-cas 9: Cleaved-caspase 9; Cleaved-cas 3: Cleaved-caspase 3 (*P < 0.05, **P < 0.01, and ***P < 0.001, compared with the PA-treated cells; #P < 0.05, ##P < 0.01, and ###P < 0.001, compared with PA+TSH-treated cells). (e) S-palmitoylation levels of TSHR after PA treatment. HAM: hydroxylamine; Palm: palmitoylation; IP: immunoprecipitation; IB: immunoblotting. *P < 0.05, **P < 0.01, and ***P < 0.001, compared with the BSA-treated cells. (f) TSHR expression in cell lysates. There was no significant difference of TSHR expression in cell lysates. (g) Predicted palmitoylation sites in TSHR using GPS-lipid. (h) Visualization of the predicted lipid modification sites. All data are presented as mean ± SD.