Journal: BMC Molecular Biology

Article Title: Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein

doi: 10.1186/s12867-016-0066-8

Figure Lengend Snippet: Analysis of DEspR translatability. a Schematic diagram of DEspR protein and mAb-epitopes. Two distinct peptides ( epitope - 1, epitope - 2 ) in the extracellular domain were used to develop murine monoclonal antibodies (mAbs). Two high-affinity mAbs target human-specific epitope-1: 7c5b2, 5g12e8; and one high-affinity mAb targets the pan-species reactive epitope-2, identical in human, monkey, and rat. Epitope-2 spans the putative ligand binding domain . The 5g12e8 mAb was used in pull-down experiments; 5g12e8 and 6g8g7 were used in Western blot analyses, 7c5b2 mAb was used in FACs analysis, immunostaining, and internalization assays, and all three were used in functional inhibition assays. The contested tryptophan (W)-aa#14 ( red ); consensus glycosylation site sequence: ( green , N-F-S-G), known internalization recognition sequence: ( blue : T-D-V-P). A blue arrow marks the splice junction between exon1 and exon2, i.e. between amino-acids G and K (aa#5-#6). b Sequential Western blot analyses of pull-down proteins from glioblastoma U87 membrane proteins using different antibodies specific for proteins identified by mass spectrometry analysis of pull-down protein-products. The identical blot was sequentially probed, stripped of antibody, confirmed as stripped, then re-probed in the following order: #1: anti-hDEspR-5g12e8 mouse mAb, #2: anti-Rab1b rabbit polyclonal Ab (pAb), #3: anti-Galectin-1 rabbit pAb; #4: anti-TMED10 rabbit pAb. Molecular weight markers are noted. DEspR bands are ~17.5 and 12.5 kDa. Expected sizes are detected for Rab1b: 22 kDa, Galectin-1: 14 kDa, and TMED10: 25 kDa. c Panel -1 shows silver-stained gel of pull-down protein products using 5g12e8 mAb from membrane proteins isolated from: (1) glioblastoma U87 CSCs, (2) PNGase-digested sample of pull-down proteins from U87 CSCs, (3) permanent transfectants DEspR-positive Cos1-cells. Panel -2 shows Western blot analysis using anti-DEspR 5g12e8 mAb showing DEspR band ( lane 1 ), smaller DEspR + band after PNGase digest-samples ( lane 2 ), and identically-sized DEspR band in DEspR-positive Cos1-cell permanent transfectants showing appropriate splicing and translatability of DEspR-minigene transfected into Cos-1 cells ( lane 3 ). Panel -3 Western blot analysis of different wells in the same gel run probed with anti-Galectin-1 pAb showing distinct sized protein bands, thus confirming DEspR-specific bands are not Galectin-1 protein bands, and that Galectin-1 is not glycosylated as reported. d Western blot analysis of Galectin-1 recombinant protein. Panel 1 overlay of gel-image and western blot image showing detection of Galectin-1 recombinant protein at expected size 14 kDa. Panel 2 overlay of gel-image and western blot image probed with anti-DEspR 5g12e8 mAb showing non-cross reactivity of anti-hDEspR mAb with Galectin-1. e Sequential western blot analysis of 5g12e8-pull-down proteins from U87 CSCs probed first with 6g8g7 ( left panel ), and subsequently with 5g12e8 after ‘stripping’ ( right panel ), detects identical protein bands. This confirms that 6g8g7 epitope is on the same protein as 5g12e8 epitope, thus corroborating DEspR protein existence

Article Snippet: Verified glioblastoma U87 MG (cat# ATCC HTB-14), triple negative breast cancer MDA-MB-231 (cat# ATCC HTB-26), non-small cell lung cancer NCI-H460 (cat# ATCC HTB-177) and pancreatic cancer Panc-1 (cat# ATCC CRL-1469) cell lines were obtained from ATCC.

Techniques: Bioprocessing, Ligand Binding Assay, Western Blot, Immunostaining, Functional Assay, Inhibition, Glycoproteomics, Sequencing, Membrane, Mass Spectrometry, Molecular Weight, Staining, Isolation, Transfection, Recombinant, Stripping Membranes

Journal: BMC Molecular Biology

Article Title: Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein

doi: 10.1186/s12867-016-0066-8

Figure Lengend Snippet: Proteins pulled-down with anti-DEspR mAb 5g12e8 from U87 CSC membranes

Article Snippet: Verified glioblastoma U87 MG (cat# ATCC HTB-14), triple negative breast cancer MDA-MB-231 (cat# ATCC HTB-26), non-small cell lung cancer NCI-H460 (cat# ATCC HTB-177) and pancreatic cancer Panc-1 (cat# ATCC CRL-1469) cell lines were obtained from ATCC.

Techniques:

Journal: BMC Molecular Biology

Article Title: Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein

doi: 10.1186/s12867-016-0066-8

Figure Lengend Snippet: Dual-fluorescence co-immunostaining analysis of DEspR and Galectin-1 expression. a Analysis of the expanding tumor zone of a U87-CSC xenograft tumor invading through the tumor fibrous cap. Co-localization ( yellow , yellow dotted circle ) of increased human-DEspR expression ( red dotted circle ) in invasive U87 tumor cells and Galectin-1 ( green dotted circle ) is observed in the invasive front. {}, invasive tumor front. Host subcutaneous tissue is to the upper right corner. b Co-localization of hDEspR and Galectin-1 is detected in tumor cells adhering to the outer wall of a microvessel in the subcutaneous tissue demonstrating invasive nature of U87 in xenograft tumors and homing to microvessels

Article Snippet: Verified glioblastoma U87 MG (cat# ATCC HTB-14), triple negative breast cancer MDA-MB-231 (cat# ATCC HTB-26), non-small cell lung cancer NCI-H460 (cat# ATCC HTB-177) and pancreatic cancer Panc-1 (cat# ATCC CRL-1469) cell lines were obtained from ATCC.

Techniques: Fluorescence, Immunostaining, Expressing

Journal: BMC Molecular Biology

Article Title: Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein

doi: 10.1186/s12867-016-0066-8

Figure Lengend Snippet: Demonstration of DEspR protein and functionality in different human tumor cell lines by anti-DEspR mAbs. a FACs analysis of different cancer tissue type CSCs: glioblastoma, triple negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), and pancreatic ductal adenocarcinoma (PDAC) using AF-568 labeled murine anti-DEspR mAbs compared with AF568-IgG2b murine isotype control. In Panc1 CSCs, low and high DEspR + CSCs are detected. b Multiple murine anti-DEspR mAbs (5g12e8, 7c5b2, 6g8g7) inhibit CSC growth in suspension culture determined by the number of live CSCs after 5 days of incubation with murine anti-DEspR mAbs, compared with corresponding control non-treated CSCs. Comparative analysis is presented using % change from respective controls; ***, One Way ANOVA with Tukey’s multiple comparisons test P < 0.0001. Epitope 1 murine mAbs: 5g12, 5g12e8, 7c5, 7c5b2; Epitope 2 murine mAbs: 6g8, 6g8e8

Article Snippet: Verified glioblastoma U87 MG (cat# ATCC HTB-14), triple negative breast cancer MDA-MB-231 (cat# ATCC HTB-26), non-small cell lung cancer NCI-H460 (cat# ATCC HTB-177) and pancreatic cancer Panc-1 (cat# ATCC CRL-1469) cell lines were obtained from ATCC.

Techniques: Labeling, Control, Suspension, Incubation

Journal: Nanomaterials

Article Title: Laminin Adsorption and Adhesion of Neurons and Glial Cells on Carbon Implanted Titania Nanotube Scaffolds for Neural Implant Applications

doi: 10.3390/nano12213858

Figure Lengend Snippet: Number of viable (green) and dead (red) U87-MG and differentiated SH-SY5Y cells on pristine and C implanted TNSs after 1 and 4 day(s). Initially, 2000 U87-MG cells per square centimeter and 9000 differentiated SH-SY5Y cells per square centimeter were seeded for the one-day test (gray line). To perform the four-day test, 1000 U87-MG cells per square centimeter and 6000 differentiated SH-SY5Y cells per square centimeter were seeded (gray line). Significant differences of p < 0.05 are marked with (*), and differences of p < 0.01 are marked with (**). Ion fluences are given in ions·cm − 2 .

Article Snippet: To examine cell viability, cytotoxicity and morphology on TNSs, U87-MG Uppsala glioblastoma cells (Cat. No. 300367, CLS Cell Lines Service GmbH) and SH-SY5Y neuroblastoma cells (Cat. No. CRL-2266, ATCC LGC Standards GmbH) were used.

Techniques:

Journal: Nanomaterials

Article Title: Laminin Adsorption and Adhesion of Neurons and Glial Cells on Carbon Implanted Titania Nanotube Scaffolds for Neural Implant Applications

doi: 10.3390/nano12213858

Figure Lengend Snippet: Cell area and cell roundness of U87-MG and differentiated SH-SY5Y cells after incubation of one and four days(s), respectively, on examined TNSs. Analyzed cell count (#), mean (white line), median (white cross), and interquartile range (dark gray) of the data distribution of detected cells. Ion fluences are given in ions·cm − 2 .

Article Snippet: To examine cell viability, cytotoxicity and morphology on TNSs, U87-MG Uppsala glioblastoma cells (Cat. No. 300367, CLS Cell Lines Service GmbH) and SH-SY5Y neuroblastoma cells (Cat. No. CRL-2266, ATCC LGC Standards GmbH) were used.

Techniques: Incubation, Cell Counting

Journal: Nanomaterials

Article Title: Laminin Adsorption and Adhesion of Neurons and Glial Cells on Carbon Implanted Titania Nanotube Scaffolds for Neural Implant Applications

doi: 10.3390/nano12213858

Figure Lengend Snippet: Analyzed extensively expanded U87-MG and SH-SY5Y cells after one day on examined TNSs. U87-MG cells are stained purple and SH-SY5Y cells are labeled pink. Increasing C implantation leads to a decline in cell protrusions of U87-MG cells. SH-SY5Y cells exhibit a decrease in the number and length of cell fibers upon rising implantation of TNSs.

Article Snippet: To examine cell viability, cytotoxicity and morphology on TNSs, U87-MG Uppsala glioblastoma cells (Cat. No. 300367, CLS Cell Lines Service GmbH) and SH-SY5Y neuroblastoma cells (Cat. No. CRL-2266, ATCC LGC Standards GmbH) were used.

Techniques: Staining, Labeling

Journal: Nanomaterials

Article Title: Laminin Adsorption and Adhesion of Neurons and Glial Cells on Carbon Implanted Titania Nanotube Scaffolds for Neural Implant Applications

doi: 10.3390/nano12213858

Figure Lengend Snippet: Images of analyzed cells acquired with ESEM after four days on the examined TNSs. To highlight the cells, we stained U87-MG purple and SH-SY5Y pink. Ion fluences are given in ions·cm − 2 .

Article Snippet: To examine cell viability, cytotoxicity and morphology on TNSs, U87-MG Uppsala glioblastoma cells (Cat. No. 300367, CLS Cell Lines Service GmbH) and SH-SY5Y neuroblastoma cells (Cat. No. CRL-2266, ATCC LGC Standards GmbH) were used.

Techniques: Staining

Journal: Oncology Letters

Article Title: Anticancer effect of the oncolytic Newcastle disease virus harboring the PTEN gene on glioblastoma

doi: 10.3892/ol.2024.14752

Figure Lengend Snippet: PTEN restoration suppresses tumorigenesis in orthotopic GBM mouse through infection of rNDV. (A) Mice were orthotopically injected with U87-MG-Luc2 cells. A total of 40 days after tumor cell injection, the mice were infected with rNDV-PTEN or rNDV via i.v. injection. (B) Body weight of in vivo mouse GBM models. (C) Survival rate of orthotopic GBM mouse models following rNDV-PTEN, rNDV or PBS injections (n=5). (D) Bioluminescent images of luciferase activity. Bioluminescent images were taken with IVIS Lumina XR and analyzed using Living Image Software (n=5). (E) MRI in orthotopic GBM mouse models. Representative MRI image of orthotopic GBM mouse models, taken 50 days after tumor injection (rNDV-PTEN, rNDV or PBS was injected 5 times). These data are expressed as the fold change in expression compared with PBS injected mice. *P<0.05 vs. PBS-injected mice. PTEN, phosphatase and tensin homolog; GBM, glioblastoma; rNDV, recombinant Newcastle disease virus; MRI, magnetic resonance imaging; i.v., intravenous.

Article Snippet: Human GBM cells, U87-MG (cat no. HTB-14; GBM of unknown origin), U87-MG-luc2 (cat. no. HTB-14-LUC2; GBM of unknown origin), T98G (cat. no. CRL-1690) and CCF-STTG1 (cat. no. CRL-1718), were purchased from American Type Culture Collection (ATCC).

Techniques: Infection, Injection, In Vivo, Luciferase, Activity Assay, Software, Expressing, Recombinant, Virus, Magnetic Resonance Imaging