The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Availability of data and materials The datasets used and/or analyzed in this study may be received from the corresponding author on reasonable request. Abbreviations BcPAPPapillary thyroid carcinomaBRAFB-type Raf kinaseCD44Cluster of differentiation 44DMSODimethyl sulfoxideERKExtracellular-signal regulated kinaseRT-qPCRquantitative real-time reverse transcription – polymerase chain reactionMAPKMitogen activated protein kinaseMEKMitogen-activated protein kinaseMMPsMatrix metalloproteinasesPTCPapillary thyroid cancerRETRearranged during transfectionRhoARas homolog family member AsiRNASmall interfering RNA Authors contributions Carbaryl BC was the principal investigator; BC, DG, and JS were involved in elaborating research hypotheses, in the study design, and the interpretation of data; JS, DG and MR performed the experiments; HD carried out the Western blot analysis and immunohistochemistry; DG performed statistical and graphical analyses; JS and BC wrote the manuscript. standard errors of the mean (SEM) from three independent experiments. B. Cell cycle assay. BcPAP and TPC1 cells after siPDPN or siNeg transfection and control cells (no siRNA) were fixed, permeabilized, stained with propidium iodide (PI) solution, and then analyzed by flow cytometry. The results are presented as percentage of cells in G1, S, and G2/M phases. (ZIP 907 kb) 12885_2018_5239_MOESM1_ESM.zip (908K) GUID:?6AF9EDBD-2537-420F-98A2-5A69D0A84D0C Data Availability StatementThe datasets used and/or analyzed in this study may be received from the corresponding author on reasonable request. Abstract Background Podoplanin (PDPN) is a mucin-type transmembrane glycoprotein specific to the lymphatic system. PDPN expression has been found in various human tumors and is considered to be a marker of Gja1 cancer. We had previously shown that PDPN expression contributes to carcinogenesis in the TPC1 papillary thyroid cancer-derived cell line by enhancing cell migration and invasiveness. The aim of this study was to determine the effect of PDPN down-regulation in another thyroid cancer-derived cell line: BcPAP. Methods In order to determine the effects of PDPN on malignant features of BcPAP cells (harboring the mutated allele) and TPC1 cells (carrying the rearrangement), we silenced PDPN in these cells using small interfering RNA (siRNA). The efficacy of PDPN silencing was confirmed by qRT-PCR and Western blotting. Then, we tested the motility and invasiveness of these cells (using scratch test Carbaryl and Transwell assay), their growth capacities F(cell cycle analysis, viability, clonogenic activity) and apoptosis assays), adhesion-independent colony-formation capacities, as well as the effect of PDPN silencing on MMPs expression and activity (zymography). Results We found that PDPN-induced cell phenotype depended on the genetic background of thyroid tumor cells. PDPN down-regulation in BcPAP cells was negatively correlated with the migration and invasion, in contrast to TPC1 cells in which PDPN depletion resulted in enhanced migration and invasiveness. Moreover, our results suggest that in BcPAP cells, PDPN may be involved in the epithelial-mesenchymal transition (EMT) through regulating the expression of the ezrin, radixin and moesin (E/R/M) proteins, MMPs 9 and MMP2, remodeling of actin cytoskeleton and cellular protrusions. We also demonstrated that PDPN expression is associated with the MAPK signaling pathway. The inhibition of the MAPK pathway resulted in a decreased PDPN expression, increased E/R/M phosphorylation and reduced cell migration. Additionally, PDPN depleted BcPAP cells treated with inhibitors of MEK1/2 kinases (U0126) or of the BRAF V600E protein (PLX4720) had reduced motility, similar to that previously observed in TPC1 cells after PDPN knock-down. Conclusions Altogether, our data suggest that PDPN may play an important role in the control of invasion and migration of papillary thyroid carcinoma cells in association with the E/R/M, MMPs and MAPK kinases. Electronic supplementary material The online version of this article (10.1186/s12885-018-5239-z) contains supplementary material, which is available to authorized users. mutation, have higher PDPN expression level. This may suggest that this gain-of-function mutation may be associated with a stronger induction of PDPN expression [19]. Therefore, we extended our analyses to the BcPAP cell line harboring a mutated allele (is a common mutation that plays a crucial role in tumorigenesis and progression of PTC [32C34]. Although signaling pathways activated by and overlap, the tumors associated with each of these two alterations have unique phenotypic features, suggesting that they also may have different tumor biology [35C39]. Therefore, in the current study, we compared the function of PDPN expressed in PTC derived cell lines with different genetic background on the modulation of cell motility, migration and invasion associated with tumor progression. We demonstrate that PDPN knock-down either promote or suppress metastatic potential of thyroid cancer cell depending on the genetic background. Overall, our Carbaryl results suggest and support the role of PDPN in thyroid tumorigenesis. Methods Cell lines and cell culture We used two thyroid cell lines derived from papillary thyroid carcinoma: BcPAP (German Collection of Microorganisms and Cell Cultures) which was previously tested and authenticated by DNA analysis, and TPC1 (established by Dr. Nobuo Sato) [40] and kindly Carbaryl provided by Dr. M. Santoro, The University of Naples Federico II, Italy). These cell lines differ in their genetic background: BcPAP cells carry the mutation in the gene, while TPC1 cells harbor the rearrangement. Both cell lines were cultured in RPMI-1640.
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- (E-F) Neither full-length nor truncated mutant IKK(R286X) protein is detectable in patients (PT), siblings, and normal peripheral blood mononuclear cells (E) and EBV-transformed B cells (F) by immunoblotting analysis with anti-N- and anti-C-terminal IKK antibodies
- Indeed, the demonstration of superantigen activity has been the standard for detecting MMTV contamination in mice because PCR cannot distinguish genomic viral RNA from endogenously-expressed MMTV transcripts, and mice infected by breast milk have suboptimal neutralizing antibody responses [78,82]
- Third, N-terminal tagging of MLKL substances, making them not capable of triggering necrotic loss of life,7, 16 didn’t prevent their translocation towards the nuclei in response to TBZ (Body 1c)
- Cells were seeded in 60-mm plates and cultured to 80C90% confluence
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