Table 1.
Sequences of gene-specific primers used for quantitative real-time PCR analysis.
Fig 1.
ER stress inducer tunicamycin impairs cell viability and activates the unfolded protein response in FRTL-5 cells.
Effect of 24-h treatment of FRTL-5 thyrocytes with different concentrations of tunicamycin (TM) on: (A) cell viability, (B) mRNA and/or protein levels of the unfolded protein response (UPR) target genes activating transcription factor 4 (ATF4) 4, BCL2 associated X, apoptosis regulator (BAX), glucose-regulated protein, 78kDa (GRP78/BiP) and C/EBP homologous protein (CHOP), (C) protein levels of protein kinase RNA-like ER kinase (PERK) and p-PERK, eukaryotic initiation factor 2α (eIF2α) and p-eIF2α, and (D) splicing of X-box binding protein (XBP)-1. (A-D) Cells treated with DMSO used as vehicle served as control. (A) Bars represent cell viability relative to control (0 μg/mL TM), which was set to 100%, and are means ± SD from 3 independent experiments. (B, C) Bars represent relative mRNA or protein levels expressed as fold of control (0 μg/mL TM), which was set to 1.0, and are means ± SD from 3 independent experiments. One representative immunoblot for BAX, BiP, CHOP, PERK, p-PERK, eIF2α and p-eIF2α and β-Actin from three independent experiments is shown. (D) Representative image of XBP-1 activation by unconventional splicing in FRTL-5 cells demonstrating the unspliced (192 bp) and the spliced (s; 166 bp) XBP-1 mRNA as detected by conventional PCR followed by agarose gel electrophoresis. Actin beta (ACTB) was detected by conventional PCR and served as a loading control for agarose gel electrophoresis. *Different from control (P < 0.05).
Fig 2.
ER stress attenuates the expression of genes involved in thyroid hormone synthesis and reduces iodide uptake in FRTL-5 cells.
Effect of 24-h treatment of FRTL-5 thyrocytes with different concentrations of tunicamycin (TM) on: (A) relative mRNA levels of sodium/iodide symporter (NIS), thyroglobulin (TG) and thyroid peroxidase (TPO), (B) protein level NIS, (C) Na125I uptake in the presence and absence of the NIS-specific inhibitor KClO4, (D) protein levels of total and p-TG, and (E) relative mRNA level of TSH receptor (TSHR). (A-E) Cells treated with DMSO used as vehicle served as control. (A, B, D, E) Bars represent relative mRNA or protein levels expressed as fold of control (0 μg/mL TM), which was set to 1.0, and are means ± SD from 3 independent experiments. (C) Bars represent counts per minute/well and are means ± SD from 3 independent experiments. (D) One representative immunoblot for total TG, p-TG and β-Actin from three independent experiments is shown. *Different from control (P < 0.05).
Fig 3.
ER stress reduces the expression of critical regulators of genes involved in thyroid hormone synthesis in FRTL-5 cells.
Effect of 24-h treatment of FRTL-5 thyrocytes with different concentrations of tunicamycin (TM) on relative mRNA and protein levels of: (A) (TTF)-1, (B) forkhead box E1 (FOXE1, also named TTF-2), and (C) paired box-8 (PAX-8). (A-C) Cells treated with DMSO used as vehicle served as control. Bars represent relative mRNA or protein levels expressed as fold of control (0 μg/mL TM), which was set to 1.0, and are means ± SD from 3 independent experiments. (A-C) One representative immunoblot for TTF-1, TTF-2, PAX-8, β-Actin and vinculin is shown. *Different from control (P < 0.05).
Fig 4.
ER stress has divergent effects on SREBP-1c and SREBP-2 pathway in FRTL-5 cells.
Effect of 24-h treatment of FRTL-5 thyrocytes with different concentrations of tunicamycin (TM) on: (A) relative mRNA levels of sterol regulatory element-binding protein-1c (SREBP-1c) and its target genes fatty acid synthase (FASN) and glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), and on protein levels of precursor (p) and nuclear (n) SREBP-1, and (B) relative mRNA levels of sterol regulatory element-binding protein-2 (SREBP-2) and its target genes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and LDL receptor (LDLR) and on protein levels of precursor (p) and nuclear (n) SREBP-2. (A, B) Cells treated with DMSO used as vehicle served as control. Bars represent relative mRNA or protein levels expressed as fold of control (0 μg/mL TM), which was set to 1.0, and are means ± SD from 3 independent experiments. (A, B) One representative immunoblot for n/pSREBP-1, n/pSREBP-2 and β-Actin is shown. *Different from control (P < 0.05).