KDa, which is comparable to the size of the glycosylation mutant

KDa, which is comparable to the size of the glycosylation mutant OASIS-513. Exposure of WT and transfected cells to brefeldin A (BFA), which causes retrograde transport of protease proteins from the Golgi to the ER, caused a reduction in both glycosylated and unglyosylated forms of OASIS and increased accumulation of the cleaved forms of the protein (Figure 3C).OASIS is Required for AN-3199 site Maximal Induction of the UPR, Chondroitin Sulfate Proteoglycan MedChemExpress 3397-23-7 expression and Glioma Cell MigrationTo address whether endogenous OASIS expressed in human glioma cell lines plays a role in the ER stress response and in extracellular matrix production, we knocked-down OASIS expression using siRNA. As shown in Figure 4A, OASIS siRNA treatment efficiently knocked-down protein expression in both U373 and U87 cells. We examined the ER stress response as measured by the induction of GRP78 and GRP94 in response to TG treatment. Interestingly, the TG induced increase in GRP78 and GRP94 chaperones was blunted in the knock-down cells compared to control (Figure 4A and B). This effect was also observed with shorter TG exposure times (Figure 4C). Analysis of spliced XBP-1 mRNA (indicative of IRE1 activation) showed that the levels of spliced XBP-1 in response to TG-induced ER stresswere not affected by OASIS knock-down. Interestingly, spliced XBP-1 was also detected in U87 glioma cells in the absence of TG treatment (Figure 4D), indicating that these fast dividing cells may experience basal ER stress and activation of a mild UPR. OASIS has also been implicated in modulating extracellular matrix components including chondroitin sulfate proteoglycans [16,18] and ER stress has been shown to upregulate chondroitin sulfate levels [33]. We thus examined the effect of OASIS knockdown on chondrotin sulfate proteoglycan protein levels using an antibody that recognizes the chondrotin sulfate glycosaminoglycans by western blot and immunofluorescence analysis [34]. ER stress induced by 48 h TG treatment resulted in reduced expression of cellular CSPGs as observed by the reduced high molecular smear detected by the anti-CSPG antibody (Figure 5A) [34]. This 1655472 was more easily observed by immunofluorescence microscopy, where the CSPG staining was lower in TG treated cells (Figure 5B). Interestingly, OASIS knock-down also effectively reduced chondroitin sulfate proteoglycan expression in nonstresssed U373 and U87 cells, relative to control siRNA treated cells (Figure 5A,B). Another extracellular matrix component shown to be induced by OASIS in bone osteoblast cells is the collagen gene Col1a1 [16]. Col1a1 mRNA was induced by 16 h, but not by 48 h TG treatment (Figure 5C,D). However, induction of this gene was not affected by OASIS knock-down in U87 glioma cells (Figure 5D). Glioma tumor cells are characterized by their highly invasive and infiltrative capacity. Given that OASIS knock-down resultedOASIS in Human Glioma CellsFigure 3. Analysis of human OASIS glycosylation in U373 astrocytes. (A) Potential OASIS glycosylation sites and mutants are indicated. (B) Wild type human OASIS-FL (OASIS-WT) and mutant (y)- constructs were transfected in U373 cells and 24 h post transfection were lysed in 1 Triton X-100 lysis buffer and immunoblotted for OASIS (anti-myc) and c-tubulin (loading control). (C) U373 cells were transfected with either wild-type fulllength human OASIS (OASIS-WT) or glycosylation-defective mutant (N-A substitution in residue 513; OASIS-513y). The cells were then treated or n.KDa, which is comparable to the size of the glycosylation mutant OASIS-513. Exposure of WT and transfected cells to brefeldin A (BFA), which causes retrograde transport of protease proteins from the Golgi to the ER, caused a reduction in both glycosylated and unglyosylated forms of OASIS and increased accumulation of the cleaved forms of the protein (Figure 3C).OASIS is Required for Maximal Induction of the UPR, Chondroitin Sulfate Proteoglycan Expression and Glioma Cell MigrationTo address whether endogenous OASIS expressed in human glioma cell lines plays a role in the ER stress response and in extracellular matrix production, we knocked-down OASIS expression using siRNA. As shown in Figure 4A, OASIS siRNA treatment efficiently knocked-down protein expression in both U373 and U87 cells. We examined the ER stress response as measured by the induction of GRP78 and GRP94 in response to TG treatment. Interestingly, the TG induced increase in GRP78 and GRP94 chaperones was blunted in the knock-down cells compared to control (Figure 4A and B). This effect was also observed with shorter TG exposure times (Figure 4C). Analysis of spliced XBP-1 mRNA (indicative of IRE1 activation) showed that the levels of spliced XBP-1 in response to TG-induced ER stresswere not affected by OASIS knock-down. Interestingly, spliced XBP-1 was also detected in U87 glioma cells in the absence of TG treatment (Figure 4D), indicating that these fast dividing cells may experience basal ER stress and activation of a mild UPR. OASIS has also been implicated in modulating extracellular matrix components including chondroitin sulfate proteoglycans [16,18] and ER stress has been shown to upregulate chondroitin sulfate levels [33]. We thus examined the effect of OASIS knockdown on chondrotin sulfate proteoglycan protein levels using an antibody that recognizes the chondrotin sulfate glycosaminoglycans by western blot and immunofluorescence analysis [34]. ER stress induced by 48 h TG treatment resulted in reduced expression of cellular CSPGs as observed by the reduced high molecular smear detected by the anti-CSPG antibody (Figure 5A) [34]. This 1655472 was more easily observed by immunofluorescence microscopy, where the CSPG staining was lower in TG treated cells (Figure 5B). Interestingly, OASIS knock-down also effectively reduced chondroitin sulfate proteoglycan expression in nonstresssed U373 and U87 cells, relative to control siRNA treated cells (Figure 5A,B). Another extracellular matrix component shown to be induced by OASIS in bone osteoblast cells is the collagen gene Col1a1 [16]. Col1a1 mRNA was induced by 16 h, but not by 48 h TG treatment (Figure 5C,D). However, induction of this gene was not affected by OASIS knock-down in U87 glioma cells (Figure 5D). Glioma tumor cells are characterized by their highly invasive and infiltrative capacity. Given that OASIS knock-down resultedOASIS in Human Glioma CellsFigure 3. Analysis of human OASIS glycosylation in U373 astrocytes. (A) Potential OASIS glycosylation sites and mutants are indicated. (B) Wild type human OASIS-FL (OASIS-WT) and mutant (y)- constructs were transfected in U373 cells and 24 h post transfection were lysed in 1 Triton X-100 lysis buffer and immunoblotted for OASIS (anti-myc) and c-tubulin (loading control). (C) U373 cells were transfected with either wild-type fulllength human OASIS (OASIS-WT) or glycosylation-defective mutant (N-A substitution in residue 513; OASIS-513y). The cells were then treated or n.