Migrating band was detected in all cell lines, which is likely the unLixisenatide glycosylated form of OASIS (TM is an N-linked glycosylation inhibitor and OASIS is a glycoprotein). Although an increase in the full-length OASIS protein in response to ER stress was detected as has been observed by others [20], ER stress-induced cleavage of OASIS was noteasily observed. However, a band migrating at the expected MW for cleaved OASIS was detected in TG treated U373 cells, which have the highest level of OASIS protein expression (Figure 2A and B). The difficulty in detecting cleaved 11967625 OASIS may be due to nuclear localization of cleaved OASIS and low levels of the cleaved form. We also observed that the ER chaperones GRP78 and GRP94 are markedly elevated in response to ER stress induced by both TM and TG, indicating these human glioma cell lines mount a robust unfolded protein response to ER stress (Figure 2A, middle panel). A time course study from 0? h indicated that in U373 and U87 cells full-length OASIS protein was markedly induced by 6 to 8 h of TG treatment, while minimal induction of OASIS was observed in A172 cells (Figure 2B ). Cleaved OASIS was also detected in response to TG treatment in the U373 cells (Figure 2B, lower arrow).Human OASIS is Glycosylated at Aspargine ResidueMouse OASIS has previously been shown to be glycosylated [20]. Human OASIS has two asparagine residues in the Cterminal ER luminal domain that are potential sites for N-linked glycosylation (Figure 3A). To examine human OASIS glycosylation constructs with asparagine to alanine HIF-2��-IN-1 substitutions at aa492 and aa513 were generated and transfected into U373 cells (Figure 3A ). Mutation at asparagine (513) completely abolishedFigure 1. OASIS mRNA is expressed in human glioma cell lines. (A) RNA was isolated lines human glioma cell lines U373, A172, U87 and rat C6 glioma cell lines and OASIS cDNA was amplified by RT-PCR. An ,1.5 kbp OASIS cDNA was amplified in all cell lines. (B) Human glioma cell lines U373, A172, U87 were treated or not with thapsigargin (TG, 1 mM 18 h) or tunicamycin (TM, 2 mg/ml 18 h). Real time PCR analysis of OASIS mRNA expression relative 1655472 to cellular b-actin mRNA. Result is from N = 3 independent experiments for each cell line. Bars are SEM. doi:10.1371/journal.pone.0054060.gOASIS in Human Glioma CellsFigure 2. OASIS is a glycoprotein protein induced in some human glioma cells in response to ER stress. (A) Glioma cell lines (U373, A172, U87) and rat C6 cells were treated or not with tunicamycin (TM, 2 mg/ml 18 h) or thapsigargin (TG, 1 mM 18 h), lysed and proteins were resolved by SDS-PAGE and immunoblotted with anti-OASIS, anti-KDEL and anti-c-tubulin antibodies. Note the higher molecular size of full-length human OASIS compared to rat OASIS protein. A non-specific protein reactive with the OASIS antibody is observed in the rat C6 samples (asterisk). (B-D) Thapsigargin (TG, 1 mM) time course study (0? h) for U373 (B), A172(C) and U87 (D) was performed. Note the induction of full-length OASIS protein in U373 and U87 cells, but negligible induction in A172 cells. Appearance of the ,50 kDa cleaved form of OASIS in response to TG treatment is observed in U373 cells (B, lower arrow). Results are representative of three independent experiments. doi:10.1371/journal.pone.0054060.gthe ,80 kDa glycosylated form (Figure 3B, C), while the 492 mutant did not have any significant effect (Figure 3B). Treatment of transfected cells with TM reduced the ,80 kDa WT protein to ,70.Migrating band was detected in all cell lines, which is likely the unglycosylated form of OASIS (TM is an N-linked glycosylation inhibitor and OASIS is a glycoprotein). Although an increase in the full-length OASIS protein in response to ER stress was detected as has been observed by others [20], ER stress-induced cleavage of OASIS was noteasily observed. However, a band migrating at the expected MW for cleaved OASIS was detected in TG treated U373 cells, which have the highest level of OASIS protein expression (Figure 2A and B). The difficulty in detecting cleaved 11967625 OASIS may be due to nuclear localization of cleaved OASIS and low levels of the cleaved form. We also observed that the ER chaperones GRP78 and GRP94 are markedly elevated in response to ER stress induced by both TM and TG, indicating these human glioma cell lines mount a robust unfolded protein response to ER stress (Figure 2A, middle panel). A time course study from 0? h indicated that in U373 and U87 cells full-length OASIS protein was markedly induced by 6 to 8 h of TG treatment, while minimal induction of OASIS was observed in A172 cells (Figure 2B ). Cleaved OASIS was also detected in response to TG treatment in the U373 cells (Figure 2B, lower arrow).Human OASIS is Glycosylated at Aspargine ResidueMouse OASIS has previously been shown to be glycosylated [20]. Human OASIS has two asparagine residues in the Cterminal ER luminal domain that are potential sites for N-linked glycosylation (Figure 3A). To examine human OASIS glycosylation constructs with asparagine to alanine substitutions at aa492 and aa513 were generated and transfected into U373 cells (Figure 3A ). Mutation at asparagine (513) completely abolishedFigure 1. OASIS mRNA is expressed in human glioma cell lines. (A) RNA was isolated lines human glioma cell lines U373, A172, U87 and rat C6 glioma cell lines and OASIS cDNA was amplified by RT-PCR. An ,1.5 kbp OASIS cDNA was amplified in all cell lines. (B) Human glioma cell lines U373, A172, U87 were treated or not with thapsigargin (TG, 1 mM 18 h) or tunicamycin (TM, 2 mg/ml 18 h). Real time PCR analysis of OASIS mRNA expression relative 1655472 to cellular b-actin mRNA. Result is from N = 3 independent experiments for each cell line. Bars are SEM. doi:10.1371/journal.pone.0054060.gOASIS in Human Glioma CellsFigure 2. OASIS is a glycoprotein protein induced in some human glioma cells in response to ER stress. (A) Glioma cell lines (U373, A172, U87) and rat C6 cells were treated or not with tunicamycin (TM, 2 mg/ml 18 h) or thapsigargin (TG, 1 mM 18 h), lysed and proteins were resolved by SDS-PAGE and immunoblotted with anti-OASIS, anti-KDEL and anti-c-tubulin antibodies. Note the higher molecular size of full-length human OASIS compared to rat OASIS protein. A non-specific protein reactive with the OASIS antibody is observed in the rat C6 samples (asterisk). (B-D) Thapsigargin (TG, 1 mM) time course study (0? h) for U373 (B), A172(C) and U87 (D) was performed. Note the induction of full-length OASIS protein in U373 and U87 cells, but negligible induction in A172 cells. Appearance of the ,50 kDa cleaved form of OASIS in response to TG treatment is observed in U373 cells (B, lower arrow). Results are representative of three independent experiments. doi:10.1371/journal.pone.0054060.gthe ,80 kDa glycosylated form (Figure 3B, C), while the 492 mutant did not have any significant effect (Figure 3B). Treatment of transfected cells with TM reduced the ,80 kDa WT protein to ,70.