Endoplasmic reticulum stress inhibits expression of genes involved in thyroid hormone synthesis and their key transcriptional regulators in FRTL-5 thyrocytes
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).