span the full length of the molecule and include surface accessible and protease sensitive loops that do not mark domain boundaries

0.05 being considered significant. Results Generation of ADAM17-silenced MC38CEA cell lines In order to study the potential role of ADAM17 in the growth and progression of colon tumors we generated several MC38CEA colon carcinoma cell lines with stably silenced ADAM17 expression via transfection with ADAM17 shRNA coding vector. Parallelly, we obtained MC38CEA cell lines stably transfected with the equivalent vector coding for non-interfering control RNA. In our experiments we used three mock-transfected cell lines that showed ADAM17 mRNA levels similar to that of the wild-type cells and three cell lines with strong silencing of ADAM17 . All ADAM17 in Tumor Development lines that continue growth upon serum deprivation, and indicates that autocrine stimulation does not play a major role in growth of MC38CEA cells in culture. Moreover, in both cases the total mitochondrial activity, which is indicative of the number of viable cells, was the same for all mock-transfected 23727046 and ADAM17-silenced cell lines after 1, 3, and 5 days in culture, although deleterious effects of the lack of serum were evident in some mock-transfected as well as ADAM17silenced cultures on day 5. The results indicate that ADAM17 does not influence proliferation of MC38CEA cells in vitro, and suggest that ADAM17 does not promote tumor development via activation of growth factors. MC38CEA cells do not express EGFR or ErbB4 ADAM17-silencing was reported previously to decrease cell proliferation. We did not observe this effect in MC38CEA cells and so we tested which of the signaling components were missing. The expression of different members of EGF family and EGF receptor family was analyzed in MC38CEA cells at the mRNA level. We found that the cells did not express TGFa for which ADAM17 is the predominant sheddase. HB-EGF, although visible at the mRNA level, was not detectable at the protein level. What is more, MC38CEA cells did not express either EGFR or ErbB4, the receptors that 15976016 may respond to stimulation by TGFa, HB-EGF, and some other factors of EGF family. Moreover, the proliferation of MC38CEA was not stimulated by external EGF in contrast to control 4T1 cells. Thus we ruled out the possibility that ADAM17 promoted MC38CEA tumor growth through the activation of the growth factors for which it was identified as the main sheddase. nous Pomalidomide biological activity rmNRG-1b did not affect growth of these cells either. Since the ErbB2/ErbB3 phosphorylation is prerequisite for intracellular signaling, we analyzed the level of ErbB2 phosphorylation to determine whether in MC38CEA, NRG-1 may activate the receptor in an autocrine manner. Western blotting analysis revealed that ADAM17-silencing resulted in a decreased level of ErbB2 phosphorylation. Moreover, ADAM17-silenced cells retained sensitivity to NRG-1, as exogenous NRG-1b caused phosphorylation of ErbB2 to the level comparable with that observed for the mock-transfected cells. However, even at a very high concentration of rmNRG1b, the level of ErbB2 phosphorylation in ADAM17silenced cells did not reach the level of its phosphorylation in mock-transfected ones suggesting a diminished sensitivity of ADAM17-silenced cells to NRG-1. ADAM17-silencing decreases MC38CEA motility in vitro To evaluate a potential impact of ADAM17 silencing on the cell motile activity we analyzed the movement of individual cells of WT, mock-transfected, and ADAM17-silenced lines recorded during an 8-h period. The trajectories of the cells are shown as circular diagrams i

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