JCS017202 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. TH and TN, at the concentrations effective in inducing dedifferentiation and an epithelial-mesenchymal-like phenotype, do not appreciably trigger apoptosis in PC Cl3 cells. PC Cl3 cells were vehicle treated or treated with 0.5 µg/ml TN or 0.5 µM TH for 30 minutes, followed by 24, 48 and 72 hours in medium without TN or TH. Apoptosis was assessed by staining cell-membrane-exposed phosphatidylserine with fluorescein isothiocyanate-conjugated annexin V (Pharmingen). Samples were analyzed by flow cytometry using a FACSCalibur (Beckman Instruments, Fullerton, CA), equipped with ModFit Software. (B) Values shown (as the percentage apoptosis) represent the mean (±s.d.) of at least three independent experiments.

• Supplemental Figure S2 -

  Fig. S2. Inhibition of JNK, p38 and phosphoinositide 3-kinase/AKT signal transduction pathways does not prevent downregulation of thyroid-specific genes. Northern blot analysis of total RNA extracted from PC Cl3 cells pretreated or not for 30 minutes with different concentrations of Ly294002, SB203580 and SP600125, then vehicle treated or treated for 30 minutes with 0.5 µM TH or 0.5 µg/ml TN in the presence or absence of inhibitors, followed by 24 hours in medium without TH or TN but with inhibitors. The same filter was probed with Pax8 and TTF-2.

• Supplemental Figure S3 -

  Fig. S3. The TGF-β−Smad signaling pathway is not involved in TH- or TN-induced thyroid dedifferentiation. (A) The TGF-β type I receptor inhibitor SB431542 did not prevent TH- or TN-induced dedifferentiation. Northern blot analysis of total RNA extracted from PC Cl3 cells pretreated or not for 30 minutes with different concentrations of SB431542, then vehicle treated or treated for 30 minutes with 0.5 µM TH and 0.5 µg/ml TN in the presence or absence of inhibitor, followed by 24 hours in medium without TH or TN but with inhibitor. The filter was probed with Tg. (B) TN and TH did not activate the TGF-β-responsive reporter pSBE4-Luc. Cells were plated to ∼80% confluence in six-well plates 24 hours before transfection. Cells were washed with serum-free medium, before addition of 1 ml of a mixture of plasmid and Lipofectamine. The plasmid-Lipofectamine mixture was made by incubating 2.5 µg of luciferase reporter plasmids, pSBE4-Luc (with four copies of the Smad binding site) or pMBE6-Luc (with three copies of the mutant Smad binding site) and 0.5 µg of pRSV-βgal with 5 µl Lipofectamine 2000 and 200 µl of serum-free medium for 30 minutes at room temperature, before dilution with 800 µl serum-free medium. Cells were incubated for 5 hours at 37°C before addition of 4 ml complete medium. After 24 hours, 0.5 µM TH or 0.5 µg/ml TN were added to the medium for 30 minutes. The medium was then replaced with medium without TH or TN. TGF-β1 was added at 10 ng/ml. 24 hours later, luciferase activities were quantified by luciferase assay (Promega) and normalized for galactosidase activity (Promega).

• Supplemental Figure S4 -

  Fig. S4. The EGF receptor is involved in thyroid dedifferentiation following ER stress. Northern blot analysis of total RNA extracted from PC Cl3 cells pretreated or not for 30 minutes with different concentrations of AG1478, then vehicle treated or treated for 30 minutes with 0.5 µg/ml TN in the presence or absence of inhibitor, followed by 24 hours in medium without TN but with inhibitor. The same filter was probed with Pax8, BiP and β-actin.