Journal: Journal of Virology

Article Title: The Rabies Virus Interferon Antagonist P Protein Interacts with Activated STAT3 and Inhibits Gp130 Receptor Signaling

doi: 10.1128/JVI.00989-13

Figure Lengend Snippet: RABV P protein inhibits nuclear translocation of endogenous cytokine-activated STAT3. (A) COS-7 cells transfected to express the indicated proteins and treated at 20 h posttransfection without or with 10 ng/ml OSM or 1,000 U/ml IFN-α for 15 min were fixed and immunostained with anti-STAT3 (Santa Cruz Biotechnology, Santa Cruz, CA; catalog no. sc-482) and Alexa Fluor 568-labeled secondary (Invitrogen, Carlsbad, CA; catalog no. A11034) antibodies before analysis using a Nikon C1 confocal laser scanning microscope; CLSM images in the red and green channels were sampled sequentially. (B) Calculation of the Fn/c ratio (mean Fn/c ratio ± the standard error of the mean; n > 60) for Alexa Fluor 568-labeled STAT3 and statistical analysis (Student's t test) were performed as described in the legend to Fig. 1. Results are from a single assay representative of six independent assays (GFP and GFP-P) or two independent assays (GFP–P-CΔ30). ***, P < 0.0001; NS, not significant; No add., no addition.

Article Snippet: As for mCherry (see above), fusion of P protein or P-CΔ30 to GFP caused its exclusion from the nucleus ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig 2 caption a7 RABV P protein inhibits nuclear translocation of endogenous cytokine-activated STAT3. (A) COS-7 cells transfected to express the indicated proteins and treated at 20 h posttransfection without or with 10 ng/ml OSM or 1,000 U/ml IFN-α for 15 min were fixed and immunostained with anti-STAT3 (Santa Cruz Biotechnology, Santa Cruz, CA; catalog no. sc-482) and Alexa Fluor 568-labeled secondary (Invitrogen, Carlsbad, CA; catalog no. {"type":"entrez-nucleotide","attrs":{"text":"A11034","term_id":"489250","term_text":"A11034"}} A11034 ) antibodies before analysis using a Nikon C1 confocal laser scanning microscope; CLSM images in the red and green channels were sampled sequentially. (B) Calculation of the Fn/c ratio (mean Fn/c ratio ± the standard error of the mean; n > 60) for Alexa Fluor 568-labeled STAT3 and statistical analysis (Student's t test) were performed as described in the legend to .

Techniques: Translocation Assay, Transfection, Labeling, Laser-Scanning Microscopy

Journal: Molecules

Article Title: Glucagon and Glucose Availability Influence Metabolic Heterogeneity and Malignancy in Pancreatic Neuroendocrine Tumour (pNET) Cells: Novel Routes for Therapeutic Targeting

doi: 10.3390/molecules30132736

Figure Lengend Snippet: Glucagon-induced glucagon receptor (GCGR) signalling promotes mitogen-activated protein kinase/ extracellular signal-regulated kinase (MAPK/ERK) signalling cascade activation in pNET malignant cells under glucose deprivation and in non-malignant cells under high glucose levels. ( A ) Immunofluorescence staining of GCGR in BON-1 cells cultured in DMEM-F12 without glucose or with high glucose levels (25 mM) for 24 h and treated with different glucagon concentrations, simulating normal physiological levels (50 pg/mL) and hyperglucagonemia-compatible concentrations (250 and 500 pg/mL). In control conditions, cells were cultured without glucagon. A primary antibody against GCGR (1:500; ab75240, Abcam) was used followed by secondary goat anti-rabbit Alexa Fluor ® 488 (green) conjugated antibody (1:1000, A-11078, Invitrogen—Thermo Fisher Scientific). Nuclei were stained blue with DAPI. Images were captured at 400× magnification using an Axio Imager.Z1 microscope (Zeiss). ( B ) Quantification of GCGR expression in BON-1 ( C , D ) and QGP-1 ( E , F ) cells cultured without glucose and in high-glucose conditions, respectively. Fluorescence quantification was performed by corrected total cell fluorescence, Image J. ( G ) Immunofluorescence staining of glucagon receptor in immortalized pancreatic α-cells (α-TC1) in DMEM cultured in the same experimental conditions as in ( A ). In control conditions, cells were cultured without glucagon. ( H ) Quantification of GCGR expression in α-TC1 cells cultured in high-glucose conditions, measured by corrected total cell fluorescence. ( I ) Western blotting for pERK1/2 and total ERK1/2 detection on BON-1, QGP-1, and αTC-1 cell lines. The values are normalized for the internal control, α-tubulin. ( J ) Western blotting quantification, using Image J, of BON-1 and QGP-1, respectively. Lane 1—control without glucose and without glucagon; lane 2—medium without glucose and 250 pg/mL glucagon; lane 3—control with high glucose levels and without glucagon; and lane 4—medium-glucose levels and 250 pg/mL glucagon. All data are presented as mean ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA. ** p < 0.001, and **** p < 0.00001 indicate a significant difference compared to control.

Article Snippet: Cells were washed 3 × with 0.5% BSA-0.1% saponin-PBS ( w / v / v ) and incubated with Alexa Fluor ® 488 goat anti-rabbit (A11034, Invitrogen) secondary antibody, diluted 1: 1000 in 0.5% BSA-0.1% saponin-PBS ( w / v / v ) for 2 h at RT.

Techniques: Activation Assay, Immunofluorescence, Staining, Cell Culture, Control, Microscopy, Expressing, Fluorescence, Western Blot, Standard Deviation