Glioblastoma (GBM) are characterized by increased invasion into the surrounding normal brain tissue. N-WASP on cell distributing. hnRNPK decreased cell migration, distributing and invasion in glioma cells. Using co-immunoprecipitation we validated the interactions of hnRNPK with N-WASP and RTVP-1 in glioma cells. In addition, we found that overexpression of RTVP-1 decreased the association of N-WASP and hnRNPK. In summary, we statement that RTVP-1 regulates glioma cell distributing, migration and invasion and that these effects are mediated via conversation with N-WASP and by interfering with the inhibitory effect of hnRNPK around the function of this protein. 0.001. The effect of RTVP-1 on glioma cell invasion was also examined by matrix degradation assay using a fluorescent labeled gelatin. As offered in Physique ?Physique1C,1C, overexpression of RTVP-1 in the A172 and U251 cells significantly increased gelatin degradation as compared with the control vector (CV) cells (Figures ?(Figures1C1C FLJ25987 and ?and1D)1D) and in accordance with the results obtained for the Boyden chamber assay. Matrix degradation has been associated with the formation of podosomes and invadopodia [28]. Podosomes are precursor structures that can mature on physiological substrates AZD8186 into invadopodium-type structures that exhibit a matrix degradation activity [29] and are recognized by the co-localization of F-actin and cortactin [30]. To examine the effect of RTVP-1 on podosome formation in glioma cells, we employed A172 cells overexpressing RTVP-1 (Physique ?(Figure1A).1A). Cells were plated on fibronectin-coated plates and podosomes were recognized by staining the cells with F-actin and anti-cortactin antibodies. As offered in Figures 1E and 1F, overexpression of RTVP-1 in the A172 cells resulted in a strong induction of podosomes in these cells compared to CV cells. To analyze the effect of RTVP-1 overexpression on invadopodia expression, cells were plated on fibronectin/gelatin-GFP and were stained for F-Actin and cortactin. Invadopodia were identified as structures stained for both F-actin and cortactin that were also able to degrade the fluorescent matrix (Physique ?(Physique1G).1G). The number of the invadopodia was significantly AZD8186 higher in A172 cells overexpressing RTVP-1 as compared to CV cells (Physique ?(Physique1H1H). RTVP-1 is usually associated with N-WASP To elucidate the mechanism underlying the effects of RTVP-1 on migration and invasion by RTVP-1 AZD8186 we performed a pull-down assay using a His-tagged RTVP-1 in U87 glioma cell lysates followed by a mass spectrometry analysis (Physique ?(Figure2A).2A). We recognized the key actin regulator protein N-WASP [31] and heterogeneous nuclear ribonucleoprotein K (hnRNPK) [32] as potential interacting proteins of RTVP-1. We first examined the expression of N-WASP in normal brain and GBM specimens and found no significant differences in the expression of this protein (Physique ?(Figure2B).2B). In contrast, we found that N-WASP expression was increased in glioma cell lines compared with normal human astrocytes (Physique ?(Physique2C)2C) and in glioma stem cells (GSCs) compared with neural stem cells (NSCs) (Physique ?(Figure2D).2D). We then analyzed the conversation of RTVP-1 with N-WASP since this protein plays a major role in actin polymerization and cell migration [33]. Using reciprocal immunoprecipitation analyses, we confirmed the conversation of RTVP-1 and N-WASP in the U87 cells and the HF2609 GSCs (Physique ?(Figure2E).2E). To further validate this conversation we performed FRET analysis AZD8186 using RTVP-1 tagged to CFP and N-WASP tagged to YFP. The two plasmids were co-transfected into U87 cells and 24 h later the cells were fixed and FRET efficiency was decided as explained in the methods. As offered in Physique ?Physique2F,2F, RTVP-1 and N-WASP showed FRET efficiency of 33.43 + 2.72%, suggesting a direct interaction of these two proteins in glioma cells. Open in a separate window Physique 2 Conversation of RTVP-1 and N-WASP in glioma cellsHis-tag affinity pull-down assay was employed as a screening assay for identifying RTVP-1 interacting proteins. The interacting complexes were resolved and stained for further analysis. N-WASP and hnRNPK were two of the pull-down complexes recognized with MassSpec analysis A. Total RNA was extracted from normal brains and GBM specimens and the expression of N-WASP was decided using real-time.