2.1. Materials

Human primary thyroid epithelial cells (Cell Biologics, Chicago, IL, USA). FTC-133 follicular thyroid cancer cells (Banca Biologica e Cell Factory, Genova, Italy). C643 anaplastic thyroid cancer cells (Dr Nils-Erik Heldin, Karolinska Institute, Stockholm, Sweden). The THJ-16T anaplastic thyroid cancer cells (Dr John Copland, Mayo Clinic, Jacksonville, FL, USA). DMEM, shRNA lentivirus particles, poly-L-lysine, BSA, fatty acid-free BSA, puromycin, HEPES, sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), RPMI 1640 (Lonza, Basel, Switzerland), F-12 Ham’s GIBCO, H6621 with supplements, gelatin coating (Cell Biologics, Chicago, IL, USA), FBS, penicillin/streptomycin, L-glutamine, trypsin–EDTA, bicinchoninic acid protein assay BCA kit, OptiMEM (Thermo Fisher Scientific, Waltham, MA, USA), Fura-2 AM (Molecular Probes, Eugene, OR, USA), Thapsigargin (Tg) (Alexis Corporation, San Diego, CA, USA), Sphingosine 1-phosphate (S1P) (Biomol International, Plymouth Meeting, PA, USA), Hsc70 antibody (Enzo Life Sciences, Inc., Farmingdale, NY, USA), TRβ1 (J51, J52), S1P3, S1P1, TPO, TG, NIS, TTF-1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Runx2 antibody, HRP-conjugated goat anti-rabbit IgG (Abcam, Waltham, MA, USA). p21waf1/cip1, p27kip1, ERK1/2, pERK1/2, β-actin, HRP-conjugated anti-rat and anti-mouse IgG antibodies (Cell Signaling Technology, Danvers, MA, USA). HRP-conjugated donkey anti-goat antibody (Promega, WI, USA). Cell culture plastic ware, human collagen type IV (Becton Dickinson, NJ, USA), Transwell inserts (Corning, NY, USA), Fura-2 AM (Molecular probes, Eugene, OR, USA), Thapsigargin (Alexis Co., San Diego, CA, USA). All chemicals used in this study were molecular biology grade.

2.5. Western Blot AnalysisThe whole cell lysates were made with lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 7 mM EDTA and 0.5% NP-40) on ice and protein concentrations were measured with the BCA protein assay kit. Then, the Laemmli sample buffer (LSB) was added to each lysate sample. Equal amounts of samples were loaded and the proteins were separated by SDS-PAGE. Next, the proteins were blotted on nitrocellulose membrane [24]. In some experiments, the proteins were blotted onto PVDF membranes using BioRad Transblot system. After blocking in 5% fat-free milk in TBS, the blots were incubated for overnight at +4 °C with respective primary antibodies TRβ1 J51 (1:200) or J52 (1:100), Runx2 (1:500), S1P1 (1:500), S1P3 (1:500), pERK1/2 (1:500), ERK1/2 (1:500), p21 (1:1000), p27 (1:1000), TPO (1:200), TTF-1 (1:200), TG (1:200), NIS (1:200) and Hsc70 (1:4000). Next day, the blots were incubated in respective secondary antibody as HRP-conjugated goat anti-rabbit (1:2000), HRP-conjugated anti-mouse (1:3000), HRP-conjugated anti rat (1:3000) and HRP-conjugated donkey anti-rabbit (1:10,000).

Proteins bands were detected with enhanced chemiluminescence (ECL; Thermo Scientific, Waltham, MA, USA). Densitometric analysis was done by using Fiji-ImageJ software. The intensities of each protein were normalized to either Hsc70 or β-Actin expression, respectively. The results are presented as expression of protein (%).