Journal: PloS one

Article Title: Suppression of uPAR retards radiation-induced invasion and migration mediated by integrin β1/FAK signaling in medulloblastoma.

doi: 10.1371/journal.pone.0013006

Figure Lengend Snippet: Figure 2. Radiation enhanced extracellular protease activity and aggressiveness of the cancer cells. (A) Medulloblastoma cells were irradiated with 7 Gy and serum starved for 24 hrs. The conditioned medium was collected and analyzed by SDS-PAGE gels either containing fibrinogen/plasminogen to determine the enzymatic activity of uPA. uPAR levels were detected in total cell lysates by immunoblotting with uPAR- specific antibodies. GAPDH antibody was used to confirm equal loading of the proteins in each lane. (B) Total RNA was isolated from the non- irradiated and irradiated cells, and transcript levels of uPA and uPAR were determined by RT-PCR analysis. (C) The enzymatic activity as determined by

Article Snippet: Cells were transfected either with plasmid-expressing siRNA against uPAR (pU) [76,77] or plasmid-expressing full length human cDNA clone of uPAR (FLuPAR) (SC319092, OriGene Technologies, Inc. Rockville, MD).

Techniques: Activity Assay, Irradiation, SDS Page, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: PloS one

Article Title: Suppression of uPAR retards radiation-induced invasion and migration mediated by integrin β1/FAK signaling in medulloblastoma.

doi: 10.1371/journal.pone.0013006

Figure Lengend Snippet: Figure 3. Radiation induced uPAR/integrin b1/FAK interaction led to activation of FAK/Src/Rac signaling. Co-immunoprecipitation assay was carried out to determine the interaction of uPAR with integrin bb1. Cells were irradiated and detached from the culture plate using a cell stripper solution. Total cell lysates were immunoprecipitated with either uPAR or integrin b1. (A) Immunoprecipitates of uPAR were analyzed by immunoblotting with integrin b1 and uPA antibodies. (B) Immunoprecipitates of uPAR/integrin b1 were analyzed by western blotting with uPAR, FAK and pFAK (Y397). (C) Immunofluorescence assay was used to detect the localization of uPAR/integrin b1 in irradiated and non-irradiated cells. Cells seeded on 2-well chamber slides were irradiated with 7 Gy and incubated for 24 hrs. The cells were incubated with uPAR and integrin b1 antibodies followed by incubation with species-specific Alexa Fluor secondary antibodies. Immunofluorescence assay demonstrates the co-localization (yellow color) of uPAR and integrin b1 at the migratory front of irradiated cells. doi:10.1371/journal.pone.0013006.g003

Article Snippet: Cells were transfected either with plasmid-expressing siRNA against uPAR (pU) [76,77] or plasmid-expressing full length human cDNA clone of uPAR (FLuPAR) (SC319092, OriGene Technologies, Inc. Rockville, MD).

Techniques: Activation Assay, Co-Immunoprecipitation Assay, Irradiation, Immunoprecipitation, Western Blot, Immunofluorescence, Incubation

Journal: PloS one

Article Title: Suppression of uPAR retards radiation-induced invasion and migration mediated by integrin β1/FAK signaling in medulloblastoma.

doi: 10.1371/journal.pone.0013006

Figure Lengend Snippet: Figure 4. uPAR overexpression enhances FAK signaling and cell invasion in medulloblastoma cells. (A) DAOY and D283 cells were transiently transfected with full length uPAR (FL-uPAR) for 48 hrs. Transfected cells were either irradiated with 7 Gy or non-irradiated and incubated for another 24 hrs. Total cell lysates were immunoblotted with the indicated antibodies. (B) Inhibiting FAK phosphorylation reduced the radiation- induced activation of FAK signaling cascade and cell invasion. DAOY and D283 cells were treated with or without FAK inhibitor for 2 hrs and then subjected to radiation and incubated for another 24 hrs. Phosphorylation levels of FAK (Y397) and downstream signal molecules, such as Paxillin and

Article Snippet: Cells were transfected either with plasmid-expressing siRNA against uPAR (pU) [76,77] or plasmid-expressing full length human cDNA clone of uPAR (FLuPAR) (SC319092, OriGene Technologies, Inc. Rockville, MD).

Techniques: Over Expression, Transfection, Irradiation, Incubation, Phospho-proteomics, Activation Assay

Journal: PloS one

Article Title: Suppression of uPAR retards radiation-induced invasion and migration mediated by integrin β1/FAK signaling in medulloblastoma.

doi: 10.1371/journal.pone.0013006

Figure Lengend Snippet: Figure 5. siRNA-mediated downregulation of uPAR reduced radiation-induced cell adhesion, invasion and migration. A plasmid expressing siRNA against the uPAR gene (pU) was transiently transfected into DAOY and D283 cells. The efficiency of uPAR downregulation was

Article Snippet: Cells were transfected either with plasmid-expressing siRNA against uPAR (pU) [76,77] or plasmid-expressing full length human cDNA clone of uPAR (FLuPAR) (SC319092, OriGene Technologies, Inc. Rockville, MD).

Techniques: Migration, Plasmid Preparation, Expressing, Transfection

Journal: PloS one

Article Title: Suppression of uPAR retards radiation-induced invasion and migration mediated by integrin β1/FAK signaling in medulloblastoma.

doi: 10.1371/journal.pone.0013006

Figure Lengend Snippet: Figure 6. Knockdown of uPAR reduced radiation-induced cell invasion and migration. Knockdown of uPAR inhibited radiation-enhanced cell adhesion, invasion and migration by blocking uPAR/integrin b1/FAK interactions. After 48 hrs of pU transfection, the cells were either irradiated or non-irradiated before analysis for cell adhesion, invasion and migration potential. Cells were detached from the culture plate using cell stripper solution for further analysis. (A) 26105 cells were plated on matrigel-coated transwell inserts and incubated for 24 hrs to determine the invasive potential of irradiated and non-irradiated pU-transfected cancer cells. The matrigel-invading cells were stained with Hema-3 and visualized under a light microscope. (B) The number of invading cells was counted from five different fields and quantified; a comparison between various treatments is represented graphically. Percent cell invasion was measured from the mean obtained from three independent experiments and values shown are the mean 6 S.D (* p,0.005 with reference to irradiated control). (C) Wound healing migration assay was carried out to determine the migratory ability of irradiated and non-irradiated pU-transfected cells. Transfected cells were grown until a monolayer formed and then a scratch was made using a 200- mL pipette tip. After thorough washing with PBS, the cells were either left non-irradiated or irradiated with 7 Gy. The distance migrated by the cells was monitored over a period of time by observation under a light microscope. (D) This was further quantified and data are represented graphically as a mean of three different experiments with 6 S.D (* represents p,0.05). doi:10.1371/journal.pone.0013006.g006

Article Snippet: Cells were transfected either with plasmid-expressing siRNA against uPAR (pU) [76,77] or plasmid-expressing full length human cDNA clone of uPAR (FLuPAR) (SC319092, OriGene Technologies, Inc. Rockville, MD).

Techniques: Knockdown, Migration, Blocking Assay, Transfection, Irradiation, Incubation, Staining, Light Microscopy, Comparison, Control, Transferring

Journal: PloS one

Article Title: Suppression of uPAR retards radiation-induced invasion and migration mediated by integrin β1/FAK signaling in medulloblastoma.

doi: 10.1371/journal.pone.0013006

Figure Lengend Snippet: Figure 7. uPAR knockdown reduces radiation-induced FAK signaling cascade by disrupting the interaction of focal adhesion molecules. (A) Total cell lysates extracted from uPAR knockdown cells (either irradiated or non-irradiated) were analyzed by immunoblotting with integrin b1, phospho FAK (Y397), Paxillin, Rac-1/Cdc42 and p130Cas. GAPDH was used as a loading control. Radiation enhanced tyrosine phosphorylation of FAK was reduced by inhibiting the interaction of uPAR and integrin b1 in pU-transfected cells. Total cell lysates were subjected to immunoprecipitation with either integrin b1 or FAK. (B) The immunoprecipitates of integrin b1 were analyzed by western blotting with antibodies specific for uPAR and FAK. (C) The co-immunoprecipitates of FAK were analyzed by western blotting with antibodies specific for phospho FAK, Paxillin and p130CAS. doi:10.1371/journal.pone.0013006.g007

Article Snippet: Cells were transfected either with plasmid-expressing siRNA against uPAR (pU) [76,77] or plasmid-expressing full length human cDNA clone of uPAR (FLuPAR) (SC319092, OriGene Technologies, Inc. Rockville, MD).

Techniques: Knockdown, Irradiation, Western Blot, Control, Phospho-proteomics, Transfection, Immunoprecipitation

Journal: PloS one

Article Title: Suppression of uPAR retards radiation-induced invasion and migration mediated by integrin β1/FAK signaling in medulloblastoma.

doi: 10.1371/journal.pone.0013006

Figure Lengend Snippet: Figure 8. Effect of uPAR siRNA expressing plasmid treatment prior to radiation on pre-established intracranial tumor. (A). Hematoxylin and eosin (H&E) staining performed on the paraffin embedded tissue sections of pre-established tumors from untreated and mice treated with either pU or pSV followed by with or without radiation. Experiments were carried out on five animals in each group. (B) Tumor volumes were quantified and represented graphically. (n = 5 with mean 6 S.D; *p,0.05 with reference to non-irradiated control mice. (C) H&E staining of the brain section was carried out and representative pictures of brain sections showing the migratory fronts and metastatic bodies from control, pSV and pU (with and without radiation) treated mice are shown. doi:10.1371/journal.pone.0013006.g008

Article Snippet: Cells were transfected either with plasmid-expressing siRNA against uPAR (pU) [76,77] or plasmid-expressing full length human cDNA clone of uPAR (FLuPAR) (SC319092, OriGene Technologies, Inc. Rockville, MD).

Techniques: Expressing, Plasmid Preparation, Staining, Irradiation, Control

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Comparative study of gavage and intraperitoneal administration of gamma-oryzanol in alleviation/attenuation in a rat animal model of renal ischemia/reperfusion-induced injury

doi: 10.22038/IJBMS.2020.51276.11642

Figure Lengend Snippet: Effect of GO on MMP-2 and MMP-9 protein expression in rats with I/R. Representative images of Western blot results of MMP-2 and MMP-9. The levels of MMP-2 (B) and MMP-9 (C)in renal tissues of rats. *** P <0.001 compared with sham control, ## P <0.01 compared with I/R. All values are mean±SEM (n=3)

Article Snippet: After that, the membranes were incubated overnight with primary antibodies against targeted proteins as follow: Bcl-2 associated x protein (BAX) (Santa Cruz Biotechnology, Inc., B-9, Mouse, Monoclonal,sc-7480), B-cell lymphoma 2 (BCL2) (N-19, sc-492 Santa Cruz Biotechnology), caspase 3 (SC-7272, Santa Cruz Biotechnology, Santa Cruz, CA), Cleaved caspase (SC-56052-Santa Cruz, CA), IL1-B (sc-32294, Santa Cruz Biotechnology), IL-6 (E-4; sc-28343; dil. 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), tumor necrosis factor-alpha ( TNF-α) (4E1]:sc-130349 Santa Cruz biotechnology, In), p- NFkB (Abcam, No: ab16502, Dilution: 1:250), p-IKB1 (Elabscience Biotechnology), MMP2 (H-76; mouse monoclonal antibody against human MMP-2, sc-10736, Santa Cruz Biotechnology, USA) and β-actin (1: 10,000, sc-47778, Santa Cruz Biotechnology Inc).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture

doi: 10.3389/fbioe.2023.1193454

Figure Lengend Snippet: Optimization of GSH chromatography wash buffer NaCl concentration and pH conditions with 34°C infection temperature CB. (A) Total viral particle distribution by anti-VP1 CE Western blot in a NaCl wash gradient from 150–1,000 mM NaCl (dotted black line) at pH 7 (green square), pH 8 (yellow circle), and pH 9 (red diamond). (B) BSA impurity clearance by BSA ELISA across GSH chromatography steps operated at pH 7 (green square), pH 8 (yellow circle), and pH 9 (red diamond).

Article Snippet: For VP1 total particle quantitation, anti-VP1 Western chemiluminescence peak areas were calibrated against a VP1 fragment (N-term 1–76, LifeTein) standard with a defined particle concentration.

Techniques: Chromatography, Concentration Assay, Infection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture

doi: 10.3389/fbioe.2023.1193454

Figure Lengend Snippet: Optimization of GSH chromatography elution buffer GSH and NaCl concentration at pH 8. (A) Analysis of GSH elution gradient fractions from 100–600 µM GSH for 37°C infection temperature harvests: microcarrier CB produced with serum at infection, total viral particle distribution by anti-VP1 CE Western blot (green circle), and empty:full capsid ratio by anti-VP4 CE Western blot (purple circle); iCELLis CB produced without serum at infection, total viral particle distribution (blue diamond), and empty:full capsid ratio (yellow diamond). (B) Analysis of GSH elution gradient fractions from 0–125 mM NaCl: 37°C infection temperature material total viral particle distribution (green circle) and % empty capsid (purple circle); 34°C infection temperature material total viral particle distribution by anti-VP1 CE Western blot (blue diamond) and % empty capsid by RP-UPLC (yellow diamond).

Article Snippet: For VP1 total particle quantitation, anti-VP1 Western chemiluminescence peak areas were calibrated against a VP1 fragment (N-term 1–76, LifeTein) standard with a defined particle concentration.

Techniques: Chromatography, Concentration Assay, Infection, Produced, Western Blot

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture

doi: 10.3389/fbioe.2023.1193454

Figure Lengend Snippet: Characterization of GSH chromatography elution purified from large-scale microcarrier bioreactors controlled at 37°C (yellow, horizontal bar) and 34°C (blue, dotted) during infection. Data represent average values from batches shown in , and error bars represent one standard deviation. (A) Relative comparison of batch productivity at GSH elution. (B) Comparison of viral genomes by RT-qPCR and viral particles by VP1 CE Western assays at GSH elution. (C) Comparison of % empty capsids by RP-UPLC in the GSH elution.

Article Snippet: For VP1 total particle quantitation, anti-VP1 Western chemiluminescence peak areas were calibrated against a VP1 fragment (N-term 1–76, LifeTein) standard with a defined particle concentration.

Techniques: Chromatography, Purification, Infection, Standard Deviation, Comparison, Quantitative RT-PCR, Western Blot