Journal: Cancer science

Article Title: Hypoxia Inhibitor Improves Iodine Uptake Disorder in Thyroid Cancer Through the hsa_circ_0023990/miR-448/DNMT1/NIS Axis.

doi: 10.1111/cas.70102

Figure Lengend Snippet: FIGURE 2 | hsa_circ_0023990 regulates NIS expression through affecting its methylation level. (A, B) The DNMT1 and NIS protein expressions in thyroid cancer tissues and paracancerous tissues were detected by western blot. (C) The NIS promoter methylation in RAIR-DTC/paracancer- ous tissues was detected by MSP assay. TPC1 cells and KMH-2 cells were transfected with hsa_circ_0023990 overexpression plasmid and si-hsa_ circ_0023990, respectively. (D–G) The DNMT1 mRNA and protein expressions in cells were detected by RT-PCR and western blot. (H–K) The NIS mRNA and protein expressions in cells were detected by RT-PCR and western blot. (L) The NIS promoter methylation in cells was detected by MSP assay. **p < 0.01, ***p < 0.001.

Article Snippet: These membranes were subsequently incubated with primary antibodies, such as anti- DNMT1 (24206- 1- AP), anti- NIS (24324- 1- AP), and GAPDH (60004- 1- lg; Proteintech) at the recommended concentration overnight at 4°C.

Techniques: Expressing, Methylation, Western Blot, MSP Assay, Transfection, Over Expression, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer science

Article Title: Hypoxia Inhibitor Improves Iodine Uptake Disorder in Thyroid Cancer Through the hsa_circ_0023990/miR-448/DNMT1/NIS Axis.

doi: 10.1111/cas.70102

Figure Lengend Snippet: FIGURE 6 | Hypoxia inhibitors increase iodine uptake in tumor tissue through the has_circ_0023990/DNMT1/NIS signaling axis to inhibit tu- mor growth. (A) H&E staining. Scale bar = 50 μm. (B, C) The cell proliferation in tumor tissues was detected by Ki-67 immunohistochemistry. “IOD/ Area” indicating the mean optical density of Ki-67 expression. Scale bar = 50 μm. (D, E) The cell apoptosis in tumor tissues was detected by TUNEL staining. Scale bar = 50 μm. (F) The hsa_circ_0023990 expression in tumor tissues was detected by RT-PCR. (G–I) The DNMT1 and NIS protein ex- pressions in tumor tissues were detected by western blot. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: These membranes were subsequently incubated with primary antibodies, such as anti- DNMT1 (24206- 1- AP), anti- NIS (24324- 1- AP), and GAPDH (60004- 1- lg; Proteintech) at the recommended concentration overnight at 4°C.

Techniques: Staining, Immunohistochemistry, Expressing, TUNEL Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Cell Stress

Article Title: Sensitive, non-immunogenic in vivo imaging of cancer metastases and immunotherapy response

doi: 10.15698/cst2023.08.288

Figure Lengend Snippet: ( A and B ) Schematic illustration of the workflow to obtain stable mNIS expression in transduced tumor cell lines without additional expression of potentially immunogenic in vitro selection markers. (C) Flow cytometry of expanded monoclonal sgKRT19 and sgScramble cancer cells prior to transduction (Ctrl), then before (Pre) and after (Post) adeno-Flpo transduction. (D) Western blot analysis for mNIS and KRT19 expression in monoclonal sgKRT19 and sgScramble cells following Ad-Flpo transfection and selection for the absence of GFP fluorescence (left two lanes). Blotting results on protein extracts from both non-mNIS expressing cells (NIS ctrl-) and mNIS expressing cells (NIS ctrl+) are shown in the right two lanes.

Article Snippet: Removal of the positive selection cassette was further confirmed by PCR analysis, while the preservation of mNIS expression was assessed by Western blotting with an anti-NIS antibody (1:200 dilution; #514487, Santa Cruz, Dallas, TX).

Techniques: Expressing, In Vitro, Selection, Flow Cytometry, Transduction, Western Blot, Transfection, Fluorescence

Journal: Cell Stress

Article Title: Sensitive, non-immunogenic in vivo imaging of cancer metastases and immunotherapy response

doi: 10.15698/cst2023.08.288

Figure Lengend Snippet: ( A and B ) A representative SPECT/CT maximum intensity projection (MIP) and a 2D coronal slice image of metastatic mNIS+/GH-pancreatic tumors developing predominantly in the liver, 5 weeks after tumor cell introduction via the portal vein. Note that the thyroid and salivary glands, stomach and bladder (denoted by green arrows in A ) are sites of endogenous NIS expression or probe excretion and do not represent sites of tumor development. (C) Photograph of tumors (yellow arrows) in the liver of the same mouse imaged in A and B, taken 5 days later at necropsy (also see Figures S2 and S3).

Article Snippet: Removal of the positive selection cassette was further confirmed by PCR analysis, while the preservation of mNIS expression was assessed by Western blotting with an anti-NIS antibody (1:200 dilution; #514487, Santa Cruz, Dallas, TX).

Techniques: Single Photon Emission Computed Tomography, Expressing

Journal: eBioMedicine

Article Title: Disulfiram metabolite Cu(DDC) 2 enhances radionuclide uptake in vivo revealing insights into tumoural ablation resistance

doi: 10.1016/j.ebiom.2026.106165

Figure Lengend Snippet: Copper-bound metabolite of disulfiram augments NIS activity and radioiodide u ptake. (A) Overview of rational design and reformulation drug strategies used in study. (B) RAI uptake of copper gluconate [Cu(II)]-treated 8505C–NIS and TPC-1-NIS cells alone or in combination with disulfiram (DSF) versus untreated (UT). (C) Schematic illustrating metabolic conversion of DSF to copper diethyldithiocarbamate [Cu(DDC) 2 ]. (D) RAI uptake of Cu(DDC) 2 -treated 8505C–NIS and TPC-1-NIS cells. (E) Same as (D) but using parental 8505C and TPC-1 cells. (F) Western blot analysis of NIS expression in Cu(DDC) 2 -treated 8505C–NIS and TPC-1-NIS cells; HE, higher exposure. (G and H) Confocal imaging of 8505C–NIS-HA (G) and TPC-1-NIS-HA (H) cells treated with Cu(DDC) 2 or vehicle (DMSO). Confocal images represent HA expression (green), NIS expression (red), and a merged image (yellow). Arrows (white) indicate regions of greater NIS plasma membrane localisation; HA, haemagglutinin. Scale bar: 20 μm. (I) Western blot analysis of NIS protein levels at the PM relative to Na+/K + ATPase following the cell-surface biotinylation assay (CSBA) in 8505C–NIS cells after Cu(DDC) 2 treatment. Control: Biotin tag omitted ( upper ), total protein before biotin separation ( lower ). (J) RAI uptake of Cu(DDC) 2 -treated human primary thyrocytes. Data presented as mean ± SEM (n = 3); one-way ANOVA, Dunnett's or Tukey's post hoc test (ns, not significant; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001); unpaired two-tailed t-test ( # P < 0.05).

Article Snippet: Western blotting and cell surface biotinylation assays (CSBA) were performed as described previously., , Blots were probed with specific antibodies against Na,K-ATPase (1:1000; Cell Signalling Technology), NIS (1:1000; Proteintech), NPL4 (1:500; Cell Signalling Technology), VCP (1:1000; Cell Signalling Technology) and β-actin (1:10000; Sigma–Aldrich).

Techniques: Activity Assay, Western Blot, Expressing, Imaging, Clinical Proteomics, Membrane, Cell Surface Biotinylation Assay, Control, Two Tailed Test

Journal: eBioMedicine

Article Title: Disulfiram metabolite Cu(DDC) 2 enhances radionuclide uptake in vivo revealing insights into tumoural ablation resistance

doi: 10.1016/j.ebiom.2026.106165

Figure Lengend Snippet: Dual agonist effect of Cu(DDC) 2 enhances NIS activity . (A – C) RAI uptake in parental 8505C cells (A), parental SW1736 cells (B) and human primary thyrocytes (C) following PAX8-siRNA depletion and Cu(DDC) 2 treatment. CON: scrambled control siRNA. (D) Relative NIS mRNA in human primary thyrocytes following PAX8-siRNA depletion and Cu(DDC) 2 treatment. (E) Relative PAX8 mRNA in human primary thyrocytes following PAX8-siRNA depletion. (F) RAI uptake in 8505C–NIS and TPC-1-NIS cells following NPL4-or VCP-siRNA depletion and Cu(DDC) 2 treatment. (G) Western blot analysis of NIS, NPL4 and VCP in 8505C–NIS and TPC-1-NIS cells after NPL4-or VCP-siRNA depletion. (H) Same as (F) but in human primary thyrocytes. (I) NanoBiT evaluation of protein: protein interaction between NIS and VCP in living HeLa cells treated with CB5339 or Cu(DDC) 2 versus controls. (J) Schematic illustrating NanoBRET assay to monitor proximity of NIS with plasma membrane protein KRAS, as well as subcellular markers RAB5 (early endosome) and RAB11 (recycling endosome). Created with BioRender.com . Modified from Read ML et al. Clinical Cancer Research, 2024. (K and L) NanoBRET evaluation of NIS localisation at the PM (K, KRAS) or in ER-golgi (L, RAB1) in live HeLa and HEK293 cells treated with Cu(DDC) 2 . Data presented as mean ± SEM (n ≥ 3), one-way ANOVA, Dunnett's or Tukey's post hoc test (ns, not significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001), unpaired two-tailed t-test ( # P < 0.05, ## P < 0.01, ### P < 0.001).

Article Snippet: Western blotting and cell surface biotinylation assays (CSBA) were performed as described previously., , Blots were probed with specific antibodies against Na,K-ATPase (1:1000; Cell Signalling Technology), NIS (1:1000; Proteintech), NPL4 (1:500; Cell Signalling Technology), VCP (1:1000; Cell Signalling Technology) and β-actin (1:10000; Sigma–Aldrich).

Techniques: Activity Assay, Control, Western Blot, Clinical Proteomics, Membrane, Modification, Two Tailed Test

Journal: eBioMedicine

Article Title: Disulfiram metabolite Cu(DDC) 2 enhances radionuclide uptake in vivo revealing insights into tumoural ablation resistance

doi: 10.1016/j.ebiom.2026.106165

Figure Lengend Snippet: Copper-bound metabolite stimulates NIS activity to enhance radionuclide uptake in vivo . (A) Schematic illustrating albumin nanoencapsulation of Cu(DDC) 2 . Lower : RAI uptake in 8505C–NIS cells treated with Cu(DDC) 2 in DMSO or albumin nanoencapsulated (ALB). (B) Overview of in vivo study to investigate the effect of Cu(DDC) 2 given by IP (step 1) or IV routes (step 2) on thyroidal NIS function in WT BALB/c mice. (C and D) 99m TcO4 - uptake (C, n = 6–11) and relative NIS mRNA (D) in thyroid glands from Cu(DDC) 2 -ALB treated WT BALB/c mice given by IP route. Total animals used = 18). (E and F) Same as (C and D) but Cu(DDC) 2 -ALB given by IV route at indicated doses (n = 5–7). Total animals used = 21. ( G ) Pearson correlation analysis between thyroidal 99m TcO4 - uptake (FC, log 2 ) and relative NIS mRNA (FC, log 2 ) in Cu(DDC) 2 -ALB treated WT BALB/c mice as outlined (B). 95% CI (upper/lower) are shown. (H and I) Relative PAX8 and NKX2-1 mRNA in thyroids from Cu(DDC) 2 -ALB treated WT BALB/c mice given by IP (H) or IV (I) routes. (J and K) Same as (H and I) but relative TPO and TG mRNA. (L) Schematic illustrating the dual impact of Cu(DDC) 2 on NIS function to enhance RAI uptake by inducing NIS mRNA and inhibiting VCP activity. Inset —promoter/enhancer regions of TPO , TG and NIS genes with relative positions of NKX2-1 and PAX8 binding sites. (M) Representative H&E stained images of thyroid tissue from Tg-rtTA/tetO-BRAF V600E mice fed with DOX ( upper ) versus normal ( lower ) chow for 7 days. Scale bars, 100 μM. (N) 99m TcO4 - uptake in thyroid tissue in DOX chow fed Tg-rtTA/tetO-BRAF V600E mice treated with Cu(DDC) 2 -ALB given by IV route (n = 3–6). Total animals used = 9. Data presented as mean ± SEM, unpaired two-tailed t-test (ns, not significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001). Images created with BioRender.com .

Article Snippet: Western blotting and cell surface biotinylation assays (CSBA) were performed as described previously., , Blots were probed with specific antibodies against Na,K-ATPase (1:1000; Cell Signalling Technology), NIS (1:1000; Proteintech), NPL4 (1:500; Cell Signalling Technology), VCP (1:1000; Cell Signalling Technology) and β-actin (1:10000; Sigma–Aldrich).

Techniques: Activity Assay, In Vivo, Binding Assay, Staining, Two Tailed Test

Journal: eBioMedicine

Article Title: Disulfiram metabolite Cu(DDC) 2 enhances radionuclide uptake in vivo revealing insights into tumoural ablation resistance

doi: 10.1016/j.ebiom.2026.106165

Figure Lengend Snippet: Transcription factor and VCP/proteostasis genes predict recurrence risk in RAI-treated PTC . (A) Volcano plot comparing log 2 FC with q -value (-log base 10) for the THCA PTC cohort and 337 transcription factor (TF) genes. (B) Volcano plot comparing log 2 FC with q -value (-log base 10) for the GSE33630 dataset and 323 TF genes. (C) Venn diagram illustrating TF genes common to both GSE33630 and THCA PTC datasets. (D) Volcano plot illustrating log 2 FC compared to q -value (-log base 10) for disease-free survival (DFS) in the BRAF-like, RAI-treated PTC cohort and 337 TF gene panel. (E) Mean number of dysregulated TF genes stratified into high-risk group (bars; left y-axis) and recurrence rate (white crosses; right y-axis) in patient clusters 1 to 4 (n = 20–49). (F) Representative Kaplan–Meier analysis of DFS for BRAF-like, RAI treated PTC stratified into patient clusters 1 to 4, log-rank test. (G) Box and whisker plot showing NIS expression (log 2 ) in BRAF-like, RAI-treated PTC stratified into patient clusters 1 to 4, Kruskal–Wallis test followed by Dunn's post hoc test (ns, not significant, ∗∗∗ P < 0.001). (H) LASSO regression analysis used to construct a 22 TF gene riskscore classifier. LASSO coefficient plot (loglambda). Y-axis: coefficient value; x-axis (lower): log(λ) value, and x-axis (upper): gene number. (I – K) ROC analysis (I) and Kaplan–Meier curve of the 22 TF gene riskscore classifier in BRAF-like, RAI-treated PTC (J) or RAI-treated PTC (K). (L) Kaplan–Meier analysis of DFS in BRAF-like, RAI-treated PTC ( left ) or RAI-treated PTC ( right ) stratified with the dual TF + VCP riskscore classifier. (M) Uni- ( left ) and multivariate analysis ( right ) of RAI-treated PTC (n = 211) stratified with the dual TF + VCP classifier, adjusting for the covariates age, sex, disease stage and ATA risk group in the multivariate model. (N) Same as (M) but with the entire TCGA THCA cohort (n = 399).

Article Snippet: Western blotting and cell surface biotinylation assays (CSBA) were performed as described previously., , Blots were probed with specific antibodies against Na,K-ATPase (1:1000; Cell Signalling Technology), NIS (1:1000; Proteintech), NPL4 (1:500; Cell Signalling Technology), VCP (1:1000; Cell Signalling Technology) and β-actin (1:10000; Sigma–Aldrich).

Techniques: Whisker Assay, Expressing, Construct

Journal: Frontiers in pharmacology

Article Title: Hydrogen Sulfide Promotes Thyroid Hormone Synthesis and Secretion by Upregulating Sirtuin-1.

doi: 10.3389/fphar.2022.838248

Figure Lengend Snippet: FIGURE 4 | Effect of H2S on thyroid hormone synthesis- and secretion-related molecules in human primary thyrocytes. Changes in TPO, Pendrin, NIS, and MCT8 expression in human primary thyrocytes treated with different concentrations of NaHS by Western blot. They were upregulated in a concentration-dependent manner in NaHS.*p < .05, TPO: thyroid peroxidase, NIS: sodium/iodide symporter, MCT8: monocarboxylate transporter eight; NaHS, sodium hydrosulfide. Data are expressed as the mean ± SEM, and all experiments were performed independently three times.

Article Snippet: The NC bands were separately incubated with the following primary antibodies at 4°C overnight: NIS antibody (1:2000; Biorbyt, Cambridge, UK), TPO antibody (1:400; Santa Cruz, California, United States), Pendrin antibody (1:500; Abcam, Cambridge, UK), MCT8 antibody (1:500; Proteintech, Wuhan, China), SIRT1 antibody (1:500, Abcam), and GAPDH antibody (1:2000; TransGen Biotech, Beijing, China).

Techniques: Expressing, Western Blot, Concentration Assay

Journal: Frontiers in pharmacology

Article Title: Hydrogen Sulfide Promotes Thyroid Hormone Synthesis and Secretion by Upregulating Sirtuin-1.

doi: 10.3389/fphar.2022.838248

Figure Lengend Snippet: FIGURE 5 | The effect of H2S on thyroid hormone and related molecules in human primary thyrocytes. (A,B) SIRT1, TPO, Pendrin, NIS, MCT8 protein and mRNA expression in human primary thyrocytes stimulated with NaHS or NaHS + EX527 assessed by real-time PCR and Western blot. (C) Extracellular TPO activity of thyrocytes stimulated with NaHS or NaHS + EX527 for 48 h. Fluorescence was normalized to the corresponding protein amounts. (D) The FT4 level in supernatants of human primary thyrocytes stimulated with NaHS or NaHS + EX527 for 48 h in chemiluminescence immunoassay. Net FT4 secretion meant that FT4 values in blank culture medium without thyrocytes were subtracted from FT4 content in thyrocyte culture supernatant. *p < 0.05, **p < 0.01, ***p < 0.001. TPO: thyroid peroxidase, NIS: sodium/iodide symporter, MCT8: monocarboxylate transporter eight; NaHS, sodium hydrosulfide. Data are expressed as the mean ± SEM, and all experiments were performed independently at least three times.

Article Snippet: The NC bands were separately incubated with the following primary antibodies at 4°C overnight: NIS antibody (1:2000; Biorbyt, Cambridge, UK), TPO antibody (1:400; Santa Cruz, California, United States), Pendrin antibody (1:500; Abcam, Cambridge, UK), MCT8 antibody (1:500; Proteintech, Wuhan, China), SIRT1 antibody (1:500, Abcam), and GAPDH antibody (1:2000; TransGen Biotech, Beijing, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Fluorescence, Chemiluminescence Immunoassay

Journal: European Journal of Endocrinology

Article Title: Monocarboxylate transporter 8 deficiency: altered thyroid morphology and persistent high triiodothyronine/thyroxine ratio after thyroidectomy

doi: 10.1530/eje-11-0369

Figure Lengend Snippet: Figure 3 Histological and morphometric alterations in thyroids from Mct8-deficient mice. (A) QPCR analyses in the murine thyroid gland. Mct8 is the most abundantly expressed T3 transporter. Seven out of 14 transporters tested are shown. Liver-specific Slc10a1 was used as a negative control. Inset: Mct8 protein expression in thyroid compared with brain. Transferrin receptor (TfR) served as membrane protein control. (B) Immunohistochemistry for Mct8 in the murine thyroid. The protein is located in the basolateral membrane of thyrocytes. Thyroid tissue from an Mct8K/y mouse (right) served as negative control. Follicles are indicated by broken lines. Scale bar 50 mm. (C) Follicular cell tumour with papillary structures and nuclear features of papillary carcinoma (nuclear infoldings, grooves and other irregularities, elongation and overlapping) in Mct8-deficient thyroids as analysed by H&E staining, immunohistochemistry for thyroglobulin (Tg) and sodium/iodide symporter (NIS). Note the basolateral localisation of NIS in the aberrant epithelium. Scale bars 50 mm. (D) Follicular size is increased in Mct8-deficient thyroids. Cross-sectional follicular areas were measured, grouped according to size and the fraction of follicles falling into each group was plotted against the area. (E) Follicular epithelial extension is significantly increased in Mct8-deficient thyroids. ***P!0.001, Student’s t-test, two-sided, unpaired. (F) Activity of type I deiodinase is unaltered in Mct8-deficient thyroids. nZ6 animals/group.

Article Snippet: Immunohistochemical stainings were performed as described (11) with antibodies against Mct8 (1:250, Atlas Antibodies), thyroglobulin (Tg; 1:750, Thermo Fisher Scientific, Waltham, MD, USA) and sodium/iodide symporter (NIS; 1:1200, Acris Antibodies, Herford, Germany).

Techniques: Negative Control, Expressing, Membrane, Control, Immunohistochemistry, Staining, Activity Assay

Journal: PLoS ONE

Article Title: Resveratrol Inhibits Sodium/Iodide Symporter Gene Expression and Function in Rat Thyroid Cells

doi: 10.1371/journal.pone.0107936

Figure Lengend Snippet: Male Sprague-Dawley rats were treated with the control vehicle (Control, n = 4) or with 50 mg/kg/day resveratrol i.p. (Resv, n = 4), for 14 days. On day 15 th , the animals were sacrificed and their thyroids were removed. Immunofluorescence analysis was performed using a mouse monoclonal anti-NIS antibody and an anti-mouse fluorescein-conjugated secondary antibody, Alexa Fluor 488, (green). Po-Pro-3 iodide was used to stain the nuclei (red). The negative control was performed using a mouse IgG preparations instead of the primary antibody (data not shown). The slides were visualized under a Zeiss LSM S10 confocal microscope with a x40 immersion lens. Representative data from four experiments are showed.

Article Snippet: After this transfer, the membranes were incubated according to the manufacturer instructions, using a mouse monoclonal anti-NIS antibody (NBP1-70342, Novus Biologicals Europe, Cambridge, UK).

Techniques: Control, Immunofluorescence, Staining, Negative Control, Microscopy