By quantifying the scratch area using ImageJ v1.42l analysis software.

By quantifying the scratch area using ImageJ v1.42l analysis software.Materials and Methods Cell CultureHuman glioma cell lines U373, A172 and U87 were obtained from Dr. James Rutka (The Hospital for Sick Children, Toronto). Details for these established cell lines can be found in the following references [28,29,30,31] and the American Type Culture Collection (ATCC) (U87, HTB-14; A172, CRL-1620). The rat C6 glioma cell line was obtained from the ATCC (CCL-107). The cells were cultured and maintained in DMEM (25 mM glucose, 2 mM L-glutamine, 10 FBS, 100 U/ml penicillin, 100 mg/ml streptomycin) at 37uC with 5 CO2.Western Blot AnalysisCells were treated as described in the figure legends and washed with PBS prior to lysis in: (1 Triton X-100, 20 mM HEPES, pH 7.4, 100 mM KCl, 2 mM EDTA, 1 mM PMSF, 10 mg/ml leupeptin, and 10 mg/ml aprotinin, 10 mM NaF, 2 mM Na3VO4, and 10 nM okadaic acid) for 15?0 min on ice. The lysate was centrifuged (10 min) and protein concentration measured using the BCA protein assay kit (Pierce, Inc., Rockford, IL). Equivalent protein amounts were resolved using 10 SDSPAGE and electro-transferred to Hybond ML 281 manufacturer nitrocellulose membranes (GE Healthcare, Piscataway, NJ). Immunodetection was performed with the following primary antibodies: rabbit antiOASIS (Protein Tech Group, Inc., Chicago, IL), mouse antiKDEL, mouse anti-PDI (Stressgen Bioreagents, Victoria, BC), rabbit anti-cleaved caspase 3 (Cell Signaling), anti-c-tubulin (Sigma-Aldrich, St. Louis, MO). The secondary antibodies, antimouse HRP (GE Healthcare) and anti-rabbit HRP (Cell Signaling Technology) were used as required and detected by ECL kit (GE Healthcare, RPN2106). Immunoblots were scanned and protein intensities were quantified using Scion Image software (Frederick, MD).RT-PCR and Real-time PCR AnalysisTotal RNA was isolated from human glioma and rat C6 cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA) followed by purification using the RNeasy RNA isolation kit (Qiagen, Valencia, CA). cDNA was synthesized using the One step RTPCR kit (Qiagen) in a PTC-200 (MJ Research, Watertown, MA) thermal cycler. Real-time PCR was performed as described previously [18,32]. Briefly, total RNA was reverse transcribed to single-stranded cDNA using the High-Capacity cDNA reverse transcription kit (Applied Biosystems). The resulting cDNA was used for real time PCR analysis using the TaqMan Gene Expression system (Applied Biosystems). Primers used were from Applied Biosystems: human OASIS (Hs00369340_m1); human Col1a1 (Hs00164004_m1); human b-actin control (#4333762F).Immunocytochemistry and MicroscopyCells were treated as described in the figure legends then fixed and processed for immunofluorescence as described in reference [18]. The primary antibody used was mouse anti-chondroitin sulphate proteoglycan Cat-316 (Abcam, Cambridge, MA; ab78689). Bright-field illumination and fluorescence microscopy were performed with an 23388095 Olympus fluorescence SPDB inverted microscope (IX71) at 60X, NA 0.95 objective. Images were acquired using a CCD camera and processed using Q capture imaging software (Q imaging, Surrey, BC).Plasmid GenerationFull length rat OASIS cDNA was synthesized from rat pancreatic islet total RNA and subcloned into pCR II Topo vector (Invitrogen) as described earlier [18]. It was then ligated into the expression vector pcDNA 3.1(-) to generate pCMVrOASIS-FL (rOASIS-FL). The human OASIS expression vector (hOASIS-FL) generated as described earlier [18] was subjected.By quantifying the scratch area using ImageJ v1.42l analysis software.Materials and Methods Cell CultureHuman glioma cell lines U373, A172 and U87 were obtained from Dr. James Rutka (The Hospital for Sick Children, Toronto). Details for these established cell lines can be found in the following references [28,29,30,31] and the American Type Culture Collection (ATCC) (U87, HTB-14; A172, CRL-1620). The rat C6 glioma cell line was obtained from the ATCC (CCL-107). The cells were cultured and maintained in DMEM (25 mM glucose, 2 mM L-glutamine, 10 FBS, 100 U/ml penicillin, 100 mg/ml streptomycin) at 37uC with 5 CO2.Western Blot AnalysisCells were treated as described in the figure legends and washed with PBS prior to lysis in: (1 Triton X-100, 20 mM HEPES, pH 7.4, 100 mM KCl, 2 mM EDTA, 1 mM PMSF, 10 mg/ml leupeptin, and 10 mg/ml aprotinin, 10 mM NaF, 2 mM Na3VO4, and 10 nM okadaic acid) for 15?0 min on ice. The lysate was centrifuged (10 min) and protein concentration measured using the BCA protein assay kit (Pierce, Inc., Rockford, IL). Equivalent protein amounts were resolved using 10 SDSPAGE and electro-transferred to Hybond nitrocellulose membranes (GE Healthcare, Piscataway, NJ). Immunodetection was performed with the following primary antibodies: rabbit antiOASIS (Protein Tech Group, Inc., Chicago, IL), mouse antiKDEL, mouse anti-PDI (Stressgen Bioreagents, Victoria, BC), rabbit anti-cleaved caspase 3 (Cell Signaling), anti-c-tubulin (Sigma-Aldrich, St. Louis, MO). The secondary antibodies, antimouse HRP (GE Healthcare) and anti-rabbit HRP (Cell Signaling Technology) were used as required and detected by ECL kit (GE Healthcare, RPN2106). Immunoblots were scanned and protein intensities were quantified using Scion Image software (Frederick, MD).RT-PCR and Real-time PCR AnalysisTotal RNA was isolated from human glioma and rat C6 cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA) followed by purification using the RNeasy RNA isolation kit (Qiagen, Valencia, CA). cDNA was synthesized using the One step RTPCR kit (Qiagen) in a PTC-200 (MJ Research, Watertown, MA) thermal cycler. Real-time PCR was performed as described previously [18,32]. Briefly, total RNA was reverse transcribed to single-stranded cDNA using the High-Capacity cDNA reverse transcription kit (Applied Biosystems). The resulting cDNA was used for real time PCR analysis using the TaqMan Gene Expression system (Applied Biosystems). Primers used were from Applied Biosystems: human OASIS (Hs00369340_m1); human Col1a1 (Hs00164004_m1); human b-actin control (#4333762F).Immunocytochemistry and MicroscopyCells were treated as described in the figure legends then fixed and processed for immunofluorescence as described in reference [18]. The primary antibody used was mouse anti-chondroitin sulphate proteoglycan Cat-316 (Abcam, Cambridge, MA; ab78689). Bright-field illumination and fluorescence microscopy were performed with an 23388095 Olympus fluorescence inverted microscope (IX71) at 60X, NA 0.95 objective. Images were acquired using a CCD camera and processed using Q capture imaging software (Q imaging, Surrey, BC).Plasmid GenerationFull length rat OASIS cDNA was synthesized from rat pancreatic islet total RNA and subcloned into pCR II Topo vector (Invitrogen) as described earlier [18]. It was then ligated into the expression vector pcDNA 3.1(-) to generate pCMVrOASIS-FL (rOASIS-FL). The human OASIS expression vector (hOASIS-FL) generated as described earlier [18] was subjected.