Fig 1.
Functional diagrams of NIS and the PI3K/Akt/FoxO3a pathway.
NIS is located on the outer membrane of the thyrocyte. Its function is to pump sodium out of the follicular cells while pumping iodide into the follicular cells (A); PCB118 stimulates cells, activates the PI3K/Akt signaling pathway, and increases Akt phosphorylation levels, after which p-Akt phosphorylates its downstream target gene FoxO3a (B).
Table 1.
Primers used for quantitative real-time PCR.
Fig 2.
Effect of PCB118 on FRTL-5 cell viability and apoptosis.
Cells were treated with PCB118 at different concentrations (0.025–25000 nM in the Cell Counting Kit-8 (CCK8) assay and 0.025–25 nM in the apoptosis assay). The viability was measured using the CCK-8 assay (A). The apoptosis rate was measured using a FACSVantage SE flow cytometer (B). Data are presented as the mean ± SD of three independent experiments.*p < 0.05, compared with the DMSO control group. #p < 0.05, compared with the low PCB118-treated groups (0.025, 0.25, 2.5, and 25 nM).
Fig 3.
Exposure of FRTL-5 cells to PCB118 results in the down-regulation of NIS expression.
Cells were treated with DMSO or various concentrations of PCB118 (0.025–25 nM) for 24 or 48 h. Whole cell lysates were analyzed by western blotting using antibodies recognizing NIS (A and B). Total RNA was isolated for qRT-PCR of NIS (C), and the FRTL-5 cells were harvested for the measurement of NIS promoter activity (D). Results show sections of blots from one experiment of the three that yielded similar results (A) or represented the mean ± SD of three independent experiments (B, C, and D). GAPDH served as the loading control in western blotting.*p < 0.05, compared with the DMSO control group. The relative ratio of target mRNA/β-actin, target protein/GAPDH and promoter activity in solvent control group was set as 1.
Fig 4.
Exposure of FRTL-5 cells to PCB118 results in the up-regulation of FoxO3a or p-FoxO3a expression.
Cells were treated with DMSO or various concentrations of PCB118 (0.025–25 nM) for 24 or 48 h. Whole cell lysates were analyzed by western blotting using antibodies recognizing FoxO3a and p-FoxO3a. GAPDH served as the loading control (A and B). Total RNA was isolated for qRT-PCR of FoxO3a (C), and the PCB118-treated FRTL-5 cells were harvested for the measurement of FoxO3a promoter activity (D). Results show sections of blots from one experiment of the three that yielded similar results (A) or represent the mean ± SD of three independent experiments (B, C, and D). GAPDH served as the loading control in western blotting.*p < 0.05, compared with the DMSO control group. The relative ratio of target mRNA/β-actin, target protein/GAPDH and promoter activity in solvent control group was set as 1.
Fig 5.
FoxO3a plays a role in the down-regulation of NIS expression in PCB118-treated FRTL-5 cells.
FoxO3a was knocked down by FoxO3a-siRNA in FRTL-5 cells, and the cells were treated with PCB118 at 25 nM. Whole cell lysates were analyzed by western blotting using antibodies recognizing FoxO3a, p-FoxO3a, and NIS (A and B). FoxO3a over-expression was induced by the pcDNA3-FoxO3a plasmid, and the cells were harvested for the measurement of NIS promoter activity (C). Results show sections of blots from one experiment of the three that yielded similar results (A) or represent the mean ± SD of three independent experiments (B and C). GAPDH served as the loading control in western blotting.*p < 0.05, compared with the DMSO control group transfected with siRNA or the pcDNA3-FoxO3a plasmid; #p < 0.05, compared with the 25 nM PCB118-treated group transfected with the NC or pcDNA3 plasmid; ##p < 0.05, compared with the DMSO control group transfected with the NC or pcDNA3 plasmid. The relative ratio of target protein/GAPDH and promoter activity in solvent control group was set as 1.
Fig 6.
Akt pathway was activated in PCB118-treated FRTL-5 cells.
Cells were treated with DMSO or various concentrations of PCB118 (0.025–25 nM) for 24 or 48 h. Whole cell lysates were analyzed by western blotting using antibodies recognizing Akt and p-Akt (A and B). Total RNA was isolated for qRT-PCR of Akt (C). Up-regulation of Akt expression was induced by CA-Akt and down-regulation was induced by DN-Akt in FRTL-5 cells. After treatment with 25 nM PCB118 for 48 h, whole cell lysates were analyzed by western blotting using antibodies recognizing p-Akt, p-FoxO3a, and NIS (D and E). Results show sections of blots from one experiment of the three that yielded similar results (A and D) or represent the mean ± SD of three independent experiments (B, C, and E). GAPDH served as the loading control in western blotting. *p < 0.05, compared with the DMSO control group (Fig. 6B, C) *p < 0.05, compared with the DMSO control group transfected with the CA-Akt or DN-Akt plasmid (Fig. 6E); #p < 0.05, compared with the 25 nM PCB118-treated group transfected with the BC plasmid; ##p < 0.05, compared with the DMSO control group transfected with the BC plasmid. The relative ratio of target mRNA/β-actin and target protein/GAPDH in solvent control group was set as 1.