Figure 1.
Co-expression of Wnt/β-catenin pathway components and TTF-1 in the TPC-1 cell line and PTCs.
(A) TPC-1 cells were cultured for 24 h, total RNA was isolated, reverse transcription was performed and then cDNA was amplified by real time PCR (qRT-PCR) using primers described in Table 1. Results were compared to RPL13A expression. Data are represented as the mean of three independent experiments. (B) TPC-1 cells were incubated for 24 h, fixed and immunostained for GSK3-β, β-catenin, TCF/LEF1, TCF3-4 and TTF-1 with primary antibodies and Alexa 555 or 488-conjugated secondary antibodies. Cell nuclei were stained with TO-PRO3 iodide (blue) and fluorescence was assessed by fluorescence microscopy combined with confocal analysis. Representative images of three independent experiments are shown with magnification, ×60. (C) The expression assessment and localization of TCF-4 and TTF-1 in TPC-1 cells performed by immunocytochemical studies. TTF-1 antibody conjugated with Alexa fluor 488 (red) or TCF-4 antibody conjugated to Alexa fluor 555 (green) were used, cell nuclei were stained with TO-PRO3 iodide (blue). Fluorescence was assessed by fluorescence microscopy combined with confocal analysis (representative images of three independent experiments). (D) Immunohistochemistry experiments were carried on paraffin-embedded in papillary thyroid carcinomas (1 and 2) and in normal thyroid tissues (5 and 6) using the antibodies recognizing either TTF-1 or TCF-4, magnification ×100. Consecutive serial sections (×400) were performed for PTC tissues (3 and 4). The high power view of papillary structures corresponds to the red square on the lower magnification (×100).
Figure 2.
Silencing of Wnt components reduces TTF-1 levels and required for the basal expression of TTF-1.
A, B and C: TPC-1 cells were transfected with 50 nM of siRNAs targeting LRP6, β-catenin, LEF1 or TCF-4, and a non-targeting siRNA sequence (siNT) as control. mRNA expression of LRP6, β-catenin, LEF1, TCF4 (A) and TTF-1 (B) were analyzed by real-time PCR and normalized to RPL13A mRNA, then recorded as the fold change compared to the non targeting siRNA (siNT). A. Significant inhibition of LRP6, β-catenin, LEF1 and TCF4 was found for cells transfected with siRNAs targeting the corresponding genes compared with those transfected with the scrambled sequence. (B) Significant inhibition of TTF-1 in cells transfected with siRNAs targeting LRP6, β-catenin, LEF1 or TCF-4. (C) Western blot data showing the effect of siRNAs targeting LRP6, β-catenin, LEF1 and TCF-4 on TTF-1 protein expression, β-actin was used as internal control (images from one experiment representative of three independent experiments) (D) TPC-1 cells were co-transfected with TOP-Flash-Luc vectors or with the TTF-1-luc-2.11 and dominant-negative plasmids (LRP6Dn, DEPXDn, β-cateninDn, TCFDn) and luciferase activity was assessed 24 h later. Results were expressed as percent of transcriptional activity as described in Material and Methods section. Columns represent three independent experiments and bar the standard deviation of the mean.
Figure 3.
LiCl induces TTF-1 expression.
(A) TPC-1 cells were incubated without or with 1 mM, 5 mM, 10 mM, and 20 mM of LiCl for 10 min, 24 h and 48 h. Expression of TTF-1 mRNA was quantified by real-time PCR and normalized with RPL13A mRNA. Results of three independent experiments are reported as mean and standard deviation of the mean (SD). a: significant changes between cells cultured for 10 min and 24 h and 48 h. b: statistical significance between cells cultured for 24 h and 48 h. (B) TTF-1, β-catenin and β-actin protein levels in cells incubated for 24 h with the same LiCl concentrations; Western blot experiments were performed three times.
Figure 4.
LiCl transactivates TTF-1 promoter and induces β-catenin recruitment on TCF/LEF binding sites in TTF-1 promoter.
A: TPC-1 cells were transfected with pSV0AL-A(delta)5′ firefly luciferase reporter plasmids containing progressive deletions of the TTF-1 promoter fragments or with TOP-Flash-Luc and FOP-Flash-Luc. Cells were incubated in the absence or the presence of 10 mM LiCl for 24 h then luciferase activity was assessed. Results from three independent experiments displayed mean ± SD. p value: significant change compared with cells transfected under normoxia (Mann-Whitney U test). (B) TCF/LEF consensus sequences found in the TTF-1 promoter were localised by using the Genomatix software. (C) TPC-1 cells were incubated with or without 10 mM LiCl. ChIP was performed 1 h and 4 h later. Real time RT-PCR was achieved using β-catenin primers to detect the active promoter site between the two selected sites. p<0.05 and 0.01 and 0.001 significant change compared to control (ANOVA; Bonferroni's test). Columns, mean of at least three independent experiments; bars, SD.
Table 1.
Primers sequences used for real time-RT-PCR analysis.