Ions were measured continuously using the xCELLigence System (Roche Diagnostics). The manufacturer’s protocol was followed. The proprietary 16 well plate was used for this purpose. A background reading of the plate was taken before seeding the cells. For G179 and NHA, the wells were coated with laminin and collagen, respectively. 10,000 cells in 100 ml of media were seeded in each well and placed in the instrument for measurement. A measurement was made every 15 minutes for the next 24 hours. Each well received 1 pM of EGF-SubA or SubA in 100 ml of media or pure media. The cells were monitored for the next 96 hours and the cell proliferation was measured as a cell index and plotted against time using proprietary software. Each treatment condition was measured as quadruplets and the mean cell index is represented. Results were confirmed in at least two independent experiments.antibody for 20 minutes. The slides were counter stained with hematoxylin and detected with Ventana ChromoMap Kit. The neuropathologist confirmed the histology of all samples and was blinded to grade when determining the expression level of GRP78 in tumors.Reverse Transcriptase PCR AnalysisTotal cellular RNA was isolated using the Qiagen RNeasy kit (Qiagen, Valencia CA). Transcript level of XBP1, GRP78 and GAPDH mRNA were analyzed using 500 ng of total RNA. TaKaRa RNA PCR kit (Takara Bio USA, Madison, WI) was used for this purpose. Bip/GRP78 primer pairs: GRP78-F, 59TGCAGCAGGACATCAAGTTC-39, and GRP78-R, 59CGCTGGTCAAAGTCTTCTCC-39, amplicon size 460 bp. Xbp1 primer pairs: Xbp1-F, 59-GTTGAGAACCAGGAGTTAAGACAG-39, Xbp1-R, 59-CAGAGGGTATCTCAAGACTAGG39. Activation of Ire1 following UPR activation was measured by the splicing of mRNA encoding XBP1. A 456 bp and 430 bp PCR product is expected if the XBP1 amplicon is derived from the unspliced and spliced form, respectively. GAPDH primer pairs: GAPDH-F, 59-CTCAGACACCATGGGGAAGGTGA-39, GAPDH-R, SR-3029 web 59-ATGATCTTGAGGCTGTTGTCATA-39 amplicon size 450 bp. PCR was performed by denaturing at 94uC for 1 m, annealing at 55 uC for 1 m, elongation at 72 uC for 1 m for a total of 30 cycles, with a final extension step at 72 uC for 7 m for all amplifiable products. The PCR products were resolved in a 2.5 agarose gel and visualized under UV light.Tissue MicroarrayThe glioma tissue microarray was purchased from US Biomax (Rockville, MD; GL 103a). The slides were stained using the Ventana Discovery XT Automated system (Ventana Medical Systems, Tuscon, AZ) following the manufacturer’s protocol with proprietary reagents. The slides were deparaffinized and a heat induced antigen retrieval protocol was followed using a Ribo CC buffer (Ventana). The array was stained with rabbit anti-Bip/ GRP78 antibody (1:200; Abcam, Cambridge, MA) diluted with Dako antibody diluent (Carpenteria, CA) for 32 minutes. The slides were incubated in Ventana omniMap anti-rabbit secondaryTargeting the UPR in Glioblastoma with EGF-SubAFigure 6. EGF-SubA delays tumor growth in mice. U251 cells were injected s.c in a mouse flank model (A). When tumors reached ,150 mm3 in size, mice were randomized into two groups: vehicle control (PBS) or EGF-SubA (125 mg/kg) delivered s.c. on the stated days (arrow). To obtain a tumor growth curve, perpendicular diameter measurements of each tumor were measured with digital calipers, and LED-209 volumes were calculated using the formula (L 6 W 6 W)/2. Tumor volumes (A) and weight of mice (B) were measured every other day. Tumor volumes were normalized.Ions were measured continuously using the xCELLigence System (Roche Diagnostics). The manufacturer’s protocol was followed. The proprietary 16 well plate was used for this purpose. A background reading of the plate was taken before seeding the cells. For G179 and NHA, the wells were coated with laminin and collagen, respectively. 10,000 cells in 100 ml of media were seeded in each well and placed in the instrument for measurement. A measurement was made every 15 minutes for the next 24 hours. Each well received 1 pM of EGF-SubA or SubA in 100 ml of media or pure media. The cells were monitored for the next 96 hours and the cell proliferation was measured as a cell index and plotted against time using proprietary software. Each treatment condition was measured as quadruplets and the mean cell index is represented. Results were confirmed in at least two independent experiments.antibody for 20 minutes. The slides were counter stained with hematoxylin and detected with Ventana ChromoMap Kit. The neuropathologist confirmed the histology of all samples and was blinded to grade when determining the expression level of GRP78 in tumors.Reverse Transcriptase PCR AnalysisTotal cellular RNA was isolated using the Qiagen RNeasy kit (Qiagen, Valencia CA). Transcript level of XBP1, GRP78 and GAPDH mRNA were analyzed using 500 ng of total RNA. TaKaRa RNA PCR kit (Takara Bio USA, Madison, WI) was used for this purpose. Bip/GRP78 primer pairs: GRP78-F, 59TGCAGCAGGACATCAAGTTC-39, and GRP78-R, 59CGCTGGTCAAAGTCTTCTCC-39, amplicon size 460 bp. Xbp1 primer pairs: Xbp1-F, 59-GTTGAGAACCAGGAGTTAAGACAG-39, Xbp1-R, 59-CAGAGGGTATCTCAAGACTAGG39. Activation of Ire1 following UPR activation was measured by the splicing of mRNA encoding XBP1. A 456 bp and 430 bp PCR product is expected if the XBP1 amplicon is derived from the unspliced and spliced form, respectively. GAPDH primer pairs: GAPDH-F, 59-CTCAGACACCATGGGGAAGGTGA-39, GAPDH-R, 59-ATGATCTTGAGGCTGTTGTCATA-39 amplicon size 450 bp. PCR was performed by denaturing at 94uC for 1 m, annealing at 55 uC for 1 m, elongation at 72 uC for 1 m for a total of 30 cycles, with a final extension step at 72 uC for 7 m for all amplifiable products. The PCR products were resolved in a 2.5 agarose gel and visualized under UV light.Tissue MicroarrayThe glioma tissue microarray was purchased from US Biomax (Rockville, MD; GL 103a). The slides were stained using the Ventana Discovery XT Automated system (Ventana Medical Systems, Tuscon, AZ) following the manufacturer’s protocol with proprietary reagents. The slides were deparaffinized and a heat induced antigen retrieval protocol was followed using a Ribo CC buffer (Ventana). The array was stained with rabbit anti-Bip/ GRP78 antibody (1:200; Abcam, Cambridge, MA) diluted with Dako antibody diluent (Carpenteria, CA) for 32 minutes. The slides were incubated in Ventana omniMap anti-rabbit secondaryTargeting the UPR in Glioblastoma with EGF-SubAFigure 6. EGF-SubA delays tumor growth in mice. U251 cells were injected s.c in a mouse flank model (A). When tumors reached ,150 mm3 in size, mice were randomized into two groups: vehicle control (PBS) or EGF-SubA (125 mg/kg) delivered s.c. on the stated days (arrow). To obtain a tumor growth curve, perpendicular diameter measurements of each tumor were measured with digital calipers, and volumes were calculated using the formula (L 6 W 6 W)/2. Tumor volumes (A) and weight of mice (B) were measured every other day. Tumor volumes were normalized.
