The Effects Of The Unfolded Protein Response (UPR) In ER On Cell Adesion And Migration In HEK293 Cells In Vitro

PurposeER is a discontinuous organelle in the structure and function. It is well known that ER is not only the entrance of secretory proteins but also the intracel-lular Ca2+ store. ER changes its shape and function in adverse environmental metabolic conditions which are the so-called ER stress response. The unfolded protein response is a kind of ER stress response that is referred to as a defensive response when the misfolded and unfolded proteins are accumulated in the ER lumen. It causes the transcription of ER molecular chaperones whose products increase its capacity for protein folding and cause selective translation attenuation to decrease the protein-folding load. Recent studies have shown that UPR plays important roles in many physiological and pathological processes such as embryonic development, cell growth, cell differentiation, cell cycle regulation and the pathogenesis of many human diseases.UPR can perturb the intracellular Ca2+ homeostasis. ER molecular chaper-one Grp78 and calreticulin also have the capacity of Ca2+ binding and handling. Ca2 + is one of the second messengers in the cells whose function is closely related with cell adhesion and migration. Cell adhesion and migration is a key step in the embryonic development, inflammatory reaction, tumor invasion and metastasis. The abnormality of cell adhesion and migration can cause the malformation of the organs in the process of organogenesis . Cell adhesion and migration is a complex biological process that begins with the formation of focal adhesion/adhesion complex, lamelh'podia extension. Small GTPase RhoA plays crucial roles in the process of cell adhesion and migration by promoting the assembly of actin filaments and the formation of the stress fibers.A variety of chemical agents were used to induce the unfolded protein response. Tunicamycin that can inhibit the glycosylation of the nascent proteins in ER is widely used to induce UPR, Herein, we treated the HEK293 cells with glycosylation inhibitor Tunicamycin to induce UPR to elucidate the effects of UPR on cell adhesion and migration and the underlying mechanisms in vitro in organgenesis.Mehtods1. the induction and identification of the unfolded protein responsea. Cell culture and the induction of UPRHEK293 cells were maintained in DMEM containing 10% fetal bovine serum To induce UPR, The cells were treated with glycosylation inhibitor Tunicamycin (10ug/ml) for 6 h in 5% CO2 at 37C.b. the identification of UPRThe expression level of Grp78 was detected by Western Blot. High level expression of Grp78 was regarded as the maker of UPR.2. The effects of UPR on cell migration in HEK293 cells in vitroa. the effect of UPR on cell migration in HEK293 cells was explored using scratch wound assay.b. The ability of cell-matrix adhesion was examined using cell-matrix adhesion assay.c. The effects of UPR on the arrangements of cytoskeleton were observed u-sing fluorescent assay.d. The expression levels and phosphorylation status of Akt were detected by Western Blot.3. The effects of UPR on cell-cell interactiona. The effect of UPR on cell-cell adhesion was detected by cell dissociation assay.b. the distribution of E-cadherin in UPR cells was analyzed by immunofluo-rescence assay and compared with the untreated cells.c. The activity of E-cadherin in UPR cells was detected using detergent ex-traction assay and western blot.d. The expression level of E-cadherin was examined using western blot in UPR cells.4. The effects of UPR on cell-matrix interactiona. The effects of UPR on cell-matrix adhesion was examined by cell adhesion assay.b. The intensity of cell-matrix adhesion in UPR was explored by Shake-off assay.c. The cellular localizations of RhoA, integrinB1 and Vinculin in UPR were observed by immunofluorescence assay using untreated cells as comparison.d. The expression status of RhoA and Vinculin were measured by western blot.e. The spreading of the cells in UPR was analyzed by scanning electr...