NaI was procured from Sigma-Aldrich (cat. #409286; St. Louis, MO, USA). Cell culture media were purchased from Gibco (cat. #21700–075; Thermo Fisher Scientific, Waltham, MA, USA). Additionally, ¹³¹I-NaI was obtained from Xiai Pharmaceutical Co., Ltd. (Guangdong, China). Thyroid ¹³¹I uptake was measured using a thyroid function analyzer (Zhongke Zhongjia Science Instrument Co., Ltd., Anhui, China).

Wild-type (WT), ClC-3 overexpression (CIC-3 OE) and ClC-3 knockout (ClC-3 KO) male FVB mice (3 months old) were obtained from Cyagen Biosciences Inc. and generously provided by Professor Mao (Guangdong Pharmaceutical University) and Dr. Deng (First Affiliated Hospital of Shenzhen University), respectively. ClC-3 protein expression was analyzed following previously established methods (9). For in vivo experiments, the mice were assigned to one of two groups: the normal iodide diet group, receiving 1.5 μg/day of iodide in deionized water through intragastric administration, while the excess iodide diet group, receiving 150 μg/day of iodide in deionized water through the same administration method over the same period. These conditions were maintained for 24 h and 48 h, respectively (21). All animal experiments were approved by the Institutional Animal Care and Use Committee of Jinan University and conducted following the guidelines of the Declaration of Helsinki and national regulations. To confirm the involvement of oxidative stress, the antioxidant N-acetyl-L-cysteine (NAC), a known reactive oxygen species (ROS) inhibitor, was administered. Mice were injected intraperitoneally with a saline solution of NAC (100 mg/kg/day, Sigma-Aldrich) (22).

Immortalized rat thyroid cells (FRTL-5) were obtained from the American Type Culture Collection (ATCC). These cells were maintained in Coon’s modified F-12 M medium (Gibco, USA) supplemented with 5% calf serum and a six-hormone (6H) cocktail, including bovine thyroid-stimulating hormone (TSH; 1 mU/mL; Sigma-Aldrich, St. Louis, MO, USA), insulin (10 μg/mL; Sigma-Aldrich), hydrocortisone (0.36 ng/mL; Sigma-Aldrich), transferrin (5 μg/mL; Sigma-Aldrich), glycyl-L-histidyl-L-lysine acetate (2 ng/mL; Sigma-Aldrich), and somatostatin (10 ng/mL; Sigma-Aldrich) (23). The cells were cultured at 37°C in an atmosphere of 5% CO₂ and 95% air, with the culture medium replaced every 3–4 days. Subculturing was performed every 7 days, and cells between passages 5 and 15 were used for experiments (24).

Lentiviral-mediated transduction with HBLV-r-ClC-3shRNA-mcherry-BSD was used to knock down ClC-3 expression in FRTL-5 cells. Fluorescence imaging was performed 72 h post-transduction to assess transduction efficiency. Subsequently, protein extraction was conducted, followed by Western blot analysis to examine the effects of CIC-3 knockdown on FRTL-5 cells.

Proteins were extracted from thyroid tissues using RIPA lysis buffer. The protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). Overall, 30 μg of extracted protein was loaded onto a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel for electrophoresis. Following electrophoretic separation, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and blocked for 2 h at room temperature (RT) using 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween 20 (TBST). Subsequently, the PVDF membranes were incubated overnight at 4°C with primary antibodies at the following dilutions: CIC-3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), NIS (1:800; Proteintech, Wuhan, China), and GAPDH (1:1000; Proteintech). After primary antibody incubation, the membranes were incubated for 2 h at RT with horseradish peroxidase (HRP)-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) secondary antibodies (1:10000; Proteintech) in TBST. Excess antibodies were removed by washing the membranes three times with TBST. Protein bands were detected using a chemiluminescence imaging system. Band intensities were normalized to GAPDH as the loading control, before being quantified using ImageJ software.

Thyroid tissue sections from WT and KO mice were harvested and fixed in 4% paraformaldehyde. The sections were incubated in 3% H₂O₂ for 25 min to quench endogenous peroxidase activity. Subsequently, the sections were washed three times with PBS, each wash lasting 5 min. Blocking was performed with 3% bovine serum albumin (BSA) for 30 min at room temperature to prevent nonspecific binding. The sections were subsequently incubated overnight at 4°C with a primary antibody against ClC-3 (1:100; Cell Signaling Technology). After washing, the sections were incubated with an HRP-conjugated secondary antibody for 50 min at room temperature. Following another PBS wash, 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich) was applied to visualize antigen localization. The thyroid sections were counterstained with Mayer’s hematoxylin, washed, and mounted. Finally, the stained sections were investigated under a Nikon Eclipse E100 light microscope (25).

Frozen tissue sections from the three groups were thawed at 25°C until moisture had evaporated. Subsequently, a histochemical pen was used to outline the thyroid tissue, followed by the application of a self-fluorescence quencher for approximately 5 min. The sections were then rinsed with water for 10 min. ROS dye solution (D7008; Sigma-Aldrich) was applied to the tissue and incubated at 37°C in the dark for 30 min. The slide was permeabilized and washed three times with PBS. An anti-fluorescence quenching reagent (Servicebio G1401, Wuhan, China) was applied to the slide. The CY3-positive channel appeared red, with an excitation wavelength of 510–560 nm and an emission wavelength of 590 nm. Images were obtained with fluorescence intensity analyzed using ImageJ.

To evaluate thyroid iodine uptake, ¹³¹I uptake measurements were conducted in WT, ClC-3 OE, and ClC-3 KO mice, all maintained on a standard iodide diet. Prior to measurement, each group was intraperitoneally injected with 200 μL of saline containing 18.5 MBq of ¹³¹I, administered 2 h before the assessment. Subsequently, the mice were euthanized via cervical dislocation. For thyroid function analysis, a thyroid function analyzer (USTC Zonkia Scientific Instruments Co. Ltd., Anhui Province, China) was used by positioning the thyroid glands at a fixed distance. The percentage of ¹³¹I uptake in the thyroid was calculated using the formula (26):

Data were presented as mean values with their respective standard errors (SEs). Statistical analysis was performed using GraphPad Prism 7 software (San Diego, CA, USA). A p-value below 0.05 was considered statistically significant.

Western blot analysis was conducted to evaluate ClC-3 protein expression across three groups of mice. ClC-3 expression was highest in the OE group, lower in the WT group, and lowest in the KO group (Figure 1A). Immunohistochemical analysis (Figures 1B,C) confirmed these findings, revealing a similar expression pattern to the Western blot analysis (Figure 1A). Immunofluorescence images revealed ClC-3 expression and localization in WT, OE, and KO mice (Figures 1D,E). In WT thyroid glands, ClC-3 was predominantly localized to the basolateral and lateral membranes of thyrocytes, with minimal expression at the apical pole. ClC-3 OE mice exhibited stronger ClC-3 fluorescence (labeled with Alexa Fluor 488, green) at the basolateral and lateral membranes than the WT mice (Figure 1D). ClC-3 KO mice exhibited no detectable fluorescence, indicating the absence of ClC-3 protein expression. ¹³¹I uptake was assessed in WT, ClC-3 OE, and ClC-3 KO mice, each receiving an intraperitoneal injection of 500 μCi Na[¹³¹I]. Thyroid radioiodide uptake was evaluated after 2 h using a thyroid function analyzer. The percentages of ¹³¹I uptake in WT, ClC-3 OE, and ClC-3 KO were 13.5 ± 2.04%, 17.4 ± 4.7%, and 7.3 ± 2.4%, respectively (Figure 1F).

Immunofluorescence analyses were conducted to evaluate the subcellular localization of NIS (labeled with Cy3, red) in thyrocytes, revealing its localization in the thyroid glands of WT, ClC-3 OE, and ClC-3 KO mice. In WT mice, NIS exhibited a characteristic linear staining pattern along the basolateral and lateral membranes of thyrocytes, similar to that observed in the ClC-3 OE group (Figure 2A). The ClC-3 OE group exhibited more thyroid follicles with high NIS expression at the basal membrane than the WT group. Furthermore, in the ClC-3 OE group, NIS was detected in the cytoplasm, compared to the WT group (Figure 2A). In the ClC-3 KO group, NIS expression at the basolateral membrane of thyroid follicles was generally lower, with a significant increase in cytoplasmic localization (Figure 2A). In ClC-3 KO mice, NIS protein levels in the thyroid glands were lower than in WT and ClC-3 OE mice, while the ClC-3 OE group exhibited higher NIS protein levels than the WT group (Figure 2B). In thyroid FRTL-5 cells, ClC-3 protein expression was significantly knocked down following treatment with ClC-3 shRNA for 72 h (Figure 2C). Further analysis of the FRTL-5 cell line revealed that in the ClC-3 shRNA group, NIS was predominantly localized in the cytoplasm, while in the control group, NIS was primarily distributed at the cell membrane under normal I⁻ conditions (Figure 2D). These findings suggest that ClC-3 affects both the localization and expression of NIS under normal iodide conditions (Figures 2A–D).

To further investigate the role of ClC-3 protein in the subcellular distribution of NIS in thyrocytes, immunofluorescence analyses were conducted on thyroid glands from WT, ClC-3 OE, and ClC-3 KO mice exposed to excess iodide for 24 h and 48 h. The normal iodide diet (1.5 μg/day of iodide in deionized water) was replaced with an excess iodide diet (150 μg/day of NaI in deionized water) for the respective durations before sacrifice. In the WT group, NIS protein distribution at the lateral and basolateral poles of thyrocytes increased after 48 h compared to after 24 h. In the ClC-3 OE group, NIS was predominantly localized intracellularly at 24 h but redistributed to the basolateral and lateral membranes of thyrocytes after 48 h. In contrast, in the ClC-3 KO group, NIS remained primarily cytoplasmic in thyrocytes at both 24 h and 48 h, with no significant changes in its localization or expression (Figure 3A). NIS total fluorescence intensity was higher in the thyroids of ClC-3 OE mice than in the WT and ClC-3 KO mice after 24 h and 48 h (Figure 3B). Cytoplasmic NIS fluorescence intensity was greater in ClC-3 OE mice than in the WT and ClC-3 KO mice after 24 h under excess I⁻ administration. These findings suggest that ClC-3 may influence NIS function during iodide overexposure.

As noted earlier, NIS expression and distribution varied significantly among the three groups 24 h after iodide excess. To further investigate the role of ROS in this process, we measured its fluorescence intensity, which was highest in ClC-3 OE mice, lower in WT mice, and lowest in ClC-3 KO mice. However, under normal iodide conditions, ROS levels in thyroid tissues did not differ significantly among the three mouse groups (Figures 4A,B). Pre-treatment with the antioxidant N-acetyl-L-cysteine (L-NAC) eliminated differences in NIS distribution and fluorescence intensity, resulting in a scattered cytoplasmic NIS pattern across all groups under excess iodide (Figures 4C,D). This suggests that ClC-3 regulation of NIS function under excess iodide conditions for 24 h may be ROS-dependent.