Figure 1.
Podoplanin expression in human thyroid tissues analyzed by immunohistochemistry.
A. Immunohistochemistry detection of podoplanin was carried on paraffin-embedded tissue sections. Representative immunostaining results obtained with anti-PDPN monoclonal antibody D2-40. (a) exclusive and strong staining of lymphatic vessels in normal thyroid tissue; (b) follicular adenoma (FA) tissue negative for podoplanin; (c) follicular thyroid carcinoma (FTC) tissue negative for podoplanin; (d) papillary thyroid carcinoma (PTC) case negative for podoplanin; (e-l) intense cytoplasmic staining of PDPN in PTC cells. In e, j and i intense staining of PDPN in tumor cells, with the peritumoral margin negative for podoplanin and strong staining of lymphatic vessels as an internal positive control. Original magnification: a-100x, a-inset -100x; b-200x, b-inset -100x; c-200x; d-200x, e-100x, f-100x, g-200x; h-200x, i-100x, j-200x, k-200x, l-400x. Of 112 PTC cases, 45 (40%) displayed ectopic podoplanin expression in the cancer cells. B. Relative PDPN mRNA expression in 21 PTCs (T) and paired normal tissues (NT) analyzed by RT-qPCR. PDPN and 18S rRNA transcript levels were quantified and podoplanin expression normalized against that of the housekeeping gene (18S rRNA). Results are presented as the mean ±SEM, **P<0.001.
Table 1.
Correlation of podoplanin cellular expression with clinical and pathological parameters of human papillary carcinoma of the thyroid.
Figure 2.
Podoplanin transcript and protein expression levels in differentiated thyroid cancer derived cell lines.
A. PDPN mRNA expression in human thyroid cancer cell lines. RT-qPCR was used to evaluate levels of the transcript encoding podoplanin in total RNA prepared from NTHY control cells, PTC-derived TPC1 and BcPAP cell lines, and FTC-derived FTC133 and CGTH-W-1 cell lines. Data from four independent experiments performed in triplicate are expressed as the mean ±SEM, **P<0.001. B. Podoplanin protein levels in human thyroid cancer cell lines. Protein extracts (30 µg) were subjected to Western blot analysis with anti-PDPN monoclonal antibody D2-40 and β-actin antibody as a loading control. C. Immunofluorescence analysis of the podoplanin expression in TPC1, BcPAP, FTC133 and CGTH-W-1 thyroid cancer cell lines. PDPN was visualized by staining using anti-PDPN monoclonal antibody D2-40 followed by DyLight549-conjugated secondary antibody (red), and nuclei were counterstained with DAPI (blue). Magnification 1000x.
Figure 3.
Down-regulation of podoplanin expression in TPC1 cells following transfection with PDPN-specific siRNA.
A. RT-qPCR analysis of PDPN mRNA levels in TPC1 thyroid cancer cells 48 h after transfection with 30 nM siRNA specific for PDPN (siPDPN) or a negative control siRNA (siNEG). The results were normalized to the 18S rRNA level and bars represent the average fold change in PDPN transcript abundance in cells transfected with siPDPN compared with cells transfected with siNEG. The results are representative of four independent experiments. Data are presented as the mean ±SEM, **P<0.001. B. Western blot analysis of podoplanin and β-actin proteins in TPC1 cells before (0 h) and 48 h after transfection with siPDPN and control siNEG. C. Immunofluorescence staining of podoplanin protein in TPC1 cells transfected with siPDPN and control siNEG. Cells were stained with anti-PDPN monoclonal antibody D2-40 followed by DyLight549-conjugated secondary antibody (red), and counterstained with DAPI (blue). Magnification 1000x. D. Effect of PDPN on cell viability. D, left panel. Proliferation was measured at 24 and 48 h after transfection of TPC1 cells with siPDPN or control siNEG. Cells seeded in 96-well plates were treated with XTT mixture reagent and formazan formation was measured at 450 nm to determine the number of viable cells. Data are expressed as the mean ±SEM of at least three independent experiments performed in quintuplicate. D, right panel. Apoptosis was measured at 48 h after transfection of TPC1 cells with siPDPN or control siNEG. Cells were collected and stained with FITC Annexin V and propidium iodide, followed by flow cytometry. Representative measurements of the percentage of Annexin V+ cells are presented. Each bar represents the mean ±SEM of at least three independent experiments performed in quadruplicate. E. Function of podoplanin in cell adhesion. TPC1 cells transfected with siPDPN display adhesion capacity comparable to those of siNEG-transfected cells. Briefly, 8000 transfected cells were seeded in the wells of 96-well plates. After incubation for 24 or 48 h, the cell monolayers were washed, fixed with 4% formaldehyde for 15 min and stained with crystal violet (Merck, USA). The stained cells were lysed by treatment with 2% SDS, then the intensity of the released stain was quantified by spectrophotometry at 550 nm using a Labsystems Multiscan RC microplate reader (Thermo Fisher Scientific, Canada). Data represents three separate experiments.
Figure 4.
Silencing PDPN affects motility, migration and invasion ability of TPC1 cells.
A. Scratch wound healing assay. Representative light microscope images showing healing of wounds in monolayers of TPC1 cells transfected with siPDPN or control siNEG, at 0 and 24% FBS as a chemoattractant and after 24 h, cells that had migrated through the 8-µm pore size membrane were stained and photographed at 40x magnification. The proportion of migrating cells is presented in graph form. Data are presented as the mean ±SEM of at least three separate experiments, **P<0.001. B, right panel. The invasiveness of TPC1 cells transfected with siPDPN or control siNEG was analyzed by adding cells to a BD BioCoat Matrigel Invasion Chamber, 8-µm. The lower reservoir was filled with 10% FBS as a chemoattractant and after 24 h, cells that had moved through the Matrigel to the lower surface of the membrane were fixed, stained and the photographed at 40x magnification. The proportion of invasive cells is presented in graph form. Data are presented as the mean ±SEM of at least three separate experiments, ***P<0.0001.