Then, cells were fixed in 4% paraformaldehyde in PBS for 20?min, washed twice in 50?mm NH4Cl in PBS, and permeabilized for 5?min in 0
Then, cells were fixed in 4% paraformaldehyde in PBS for 20?min, washed twice in 50?mm NH4Cl in PBS, and permeabilized for 5?min in 0.1% Triton X-100 in PBS. by the thyrocyte) and of polarized epithelium (the single layer of thyrocytes delimiting the follicle). Following endoplasmic reticulum stress, in thyroid cells the folding of thyroglobulin was disrupted. The mRNAs of unfolded protein response were induced or spliced (X-box binding protein-1). Differentiation was inhibited: mRNA levels of thyroid specific genes, and of thyroid transcription factors were dramatically downregulated, at least in part, transcriptionally. The dedifferentiating response was accompanied by an upregulation of mRNAs of antioxidant genes. Moreover, cadherin-1, and the thyroid (and kidney)-specific cadherin-16 mRNAs were downregulated, vimentin, and SNAI1 mRNAs were upregulated. In addition, loss of cortical actin and stress fibers formation were observed. Together, these MK-0354 data indicate that ER stress in thyroid cells induces dedifferentiation, loss of epithelial organization, shift towards a mesenchymal phenotype, and activation of the antioxidant response, highlighting, at the same time, a new and wide strategy to achieve survival following ER stress, and, as a sort of the other side of the coin, a possible new molecular mechanism NOX1 of decline/loss MK-0354 of function leading to a deficit of thyroid MK-0354 hormones formation. method. Each value was first normalized to the respective value of a sample to account for variability in the concentration of RNA and in the conversion efficiency of the RT reaction. GAPDH was not affected by Th/Tn treatments. The primers used are listed in Supplementary Materials ( Table S1 ). Immunofluorescence 1.5 105 cells were plated on 12?mm diameter glass coverslips. Forty-eight hours later, cells were vehicle-treated or treated plus 0.5 g/ml Tn or 0.5 M Th for 30?min. The medium was then replaced with medium without Th/Tn and cells incubated for 24?h. Immunofluorescence was performed as previously reported (26). Briefly, cells were fixed in 4% paraformaldehyde in PBS for 20?min, washed twice in 50?mm NH4Cl in PBS, and permeabilized for 5?min in 0.1% Triton X-100 in PBS. Nuclei were stained with HOECHST 33258. Immunofluorescence analysis was performed at a confocal laser scanning microscope LSM 510 Meta (Zeiss, Gottingen, Germany). The??of diode UV laser was 405, the argon ion laser was set at 488 nm. Fluorescence emission was revealed by 420C480 band pass filter for Hoechst and by 505C530 band pass filter for Alexa Fluor 488. Double staining IF images were acquired separately in the green, and blue channels at a resolution of 1 1,024 1,024 pixels, with the confocal pinhole set to one Airy unit and then saved in TIFF format. Transient Expression Analysis Cells were plated in six-well plates to about 80% confluence 24?h before transfection. Cells were washed with serum-free medium before addition of 1 1?ml of plasmid/Lipofectamine mixture. The plasmid/Lipofectamine mixture was made by incubating 2.5 g of luciferase reporter plasmid and 0.5 g of pRL-TK vector (Promega) with 5 l Lipofectamine 2000 (Invitrogen) and 200 l of serum-free medium for 30?min at room temperature, before dilution with 800 l serum-free medium. Cells were incubated for 5?h at 37C before addition of 1 1?ml medium supplemented with 20% serum. After 24?h, cells were treated with 0.5 and 1.0 g/ml of Tn for 30?min, 1?h, and 2?h. The medium was then replaced with medium without Tn. Twenty-four hours later, firefly and renilla activities were determined in cell lysates using the Dual-Luciferase Reporter Assay System (Promega) and a luminometer (Orion I, Berthold Detection Systems) according to the manufacturers instructions. Results were expressed as the ratio of firefly to renilla activity. Western Blots Analysis Western blots were carried out as previously reported (16). Briefly, cells were treated or mock treated with Th or Tn in medium for 30?min, followed by 24?h in medium without Th/Tn. After evaluation of protein content, 30 g of cell extract was analyzed by SDS-PAGE and electrotransferred to polyvinylidene difluoride. Blocking was for 15?h at 4C with Tris-buffered saline-Tween 20 (TBST) buffer (10 mM Tris [pH 8.0], 150 mM NaCl, 0.1% Tween 20) containing 10% nonfat dry milk, followed by incubation in TBST buffer for 2?h at room temperature with a 1:2,000 dilution of anti-Tg, 1:500 anti-p-eIF2, 1:1,000 anti-ATF4/antiCDH1/antiCDH16/antiSNAI1/anti vinculin, 1:2,000 anti–actin/anti-tubulin. After being washed with TBST, the blot was incubated for 1?h at room temperature with antirabbit horseradish.