Tumor-derived cell lines display a constitutive EMT-like phenotype.

<p>qRT-PCR analysis of EMT markers (A) (E-CAD; N-CAD; CDH16; TNC; VIM; FN 1) and CDH6 (B) in non-treated thyroid-derived cell lines. The bars represent the average fold change of the indicated genes in tumor cells (B-CPAP, TPC1, WRO, and FTC-133) as compared to thyrocytes (Nthy.ori 3.1), normalized to the geometric mean of levels of three reference genes: GAPDH, CYPA, GUSB. C) Western Blot analysis of E-CAD, N-CAD, FN1 and Actin in non-treated Nthy.ori 3.1; B-CPAP and TPC1 cells. D) Western Blot analysis of phosphorylated ERK, phosphorylated AKT, and Actin in Nthy.ori 3.1 (Left panels); B-CPAP (middle panels) and TPC1 (right panels) cells untreated (NT) or after TGF-β exposure for the indicated times. E) Immunofluorescence staining of SMAD2/3 proteins (green) in Nthy.ori 3.1 (upper panels); B-CPAP (middle panels) and TPC1 (lower panels), non-treated (NT) or after TGF-β exposure for the indicated times. DAPI (Blue) stains the nuclei. Magnification 200X. F-H) qRT-PCR analysis of transcription factors known to partake in the EMT program (SNAI1, SNAI2, ZEB1, TWIST, Id1, and RUNX2) in Nthy.ori 3.1 (E), B-CPAP (F) and TPC1 (G) cells, non-treated (NT) or treated with TGF-β for the indicated times. The bars represent the fold change of the indicated genes in TGF-β treated cells as compared to the non-treated cell levels, normalized to the geometric mean of GAPDH, CYPA, GUSB levels. p-value was calculated by two-tailed Student’s t-test. *** p≤ 0.001; ** p≤ 0.01; * p≤ 0.05. Error bars represent s.e.m. (n=3).