Journal: EMBO Molecular Medicine

Article Title: Endothelial cell‐derived angiopoietin‐2 is a therapeutic target in treatment‐naive and bevacizumab‐resistant glioblastoma

doi: 10.15252/emmm.201505505

Figure Lengend Snippet: A, B Immunofluorescence staining of CD 31 and desmin in GL 261 glioblastoma after single and dual treatment with anti‐Ang‐2 ( AMG 386) and anti‐ VEGF (aflibercept) (A). Corresponding quantitative analysis of microvessel densities and pericyte numbers (B; control n = 24; AMG 386 n = 10; aflibercept n = 12; AMG 386 + aflibercept n = 13). C, D Staining for mouse IgG (red) as a surrogate parameter for vascular leakage and CD 31 (green) is shown in GL 261 glioblastoma sections following different anti‐angiogenic treatments (C) with quantification in (D); n = 3. E Quantification of necrotic areas in GL 261 glioblastoma sections after anti‐angiogenic treatment is shown as percent of whole tumor area (control, aflibercept; AMG 386 + aflibercept n = 4; AMG 386 n = 3). F Analysis of Glut1 immunoreactivity in GL 261 glioblastoma sections ( n = 3). G, H Double‐immunofluorescence stainings of macrophages (F4/80) and tumor vessels ( vWF ) in mouse GBM after treatment with AMG 386, aflibercept, or the combination of both are shown in (G). Quantitative analysis of tumor‐infiltrating leukocytes (F4/80 + , CD 3 + , Ly‐6G + ) following anti‐angiogenic treatment is displayed in (H). (Control n = 28; AMG 386 n = 12; aflibercept n = 11; AMG 386 + aflibercept n = 12). I Kaplan–Meier survival analysis of GL 261 tumors grown in C57 BL /6 mice following anti‐angiogenic treatment (control n = 38; AMG 386 n = 13; aflibercept n = 12; AMG 386 + aflibercept n = 13). J, K Double‐immunofluorescence stainings with anti‐F4/80 and anti‐ CD 206 in brain tumor sections of mice treated with anti‐Ang‐2 ( AMG 386), anti‐ VEGF (aflibercept), or the combination of both are shown in (J). Corresponding quantitative analyses of tumor‐infiltrating F4/80 + cells and CD 206 + cells, and the ratio of CD 206 + versus F4/80 + upon anti‐angiogenic therapy is displayed in (K) (control n = 21; AMG 386 n = 13; aflibercept n = 9; AMG 386 + aflibercept n = 4). L, M Flow cytometry of tumor‐infiltrating macrophages following enzymatic dissociation of mouse GL 261 brain tumors plus/minus anti‐angiogenic therapy. Percent of CD 206 + (L) and MHC class II HI cells (M) among CD 45 + CD 11b + GR 1 − F4/80 + cells is displayed (control n = 4; AMG 386 n = 4; aflibercept n = 5; AMG 386 + aflibercept n = 5). Data information: Statistical analyses were performed using one‐way ANOVA and Tukey's multiple comparison except for log‐rank and Wilcoxon (I). * P < 0.05, ** P < 0.01, *** P < 0.005, # P < 0.005 of aflibercept + AMG 386 versus aflibercept; data are mean ± SEM (A–K), mean ± SD (L, M). Scale bars (A, C, G and J): 100 μm.

Article Snippet: Anti‐mouse CD31 (clone MEC 13.1) 1:100, anti‐mouse Ly‐6G (clone 1A8) 1:100, anti‐mouse CD3e (clone 145‐2C11) 1:100 all from BD Pharmingen; anti‐mouse F4/80 (clone A3‐1) 1:100 from Biozol; goat anti‐mouse CD206 (clone C068C2) 1:100 from R&D Systems; anti‐αSMA (clone 1A4) 1:200 from Sigma; anti‐mouse Glut1 (polyclonal, C‐Terminus) 1:100 from Merck Millipore; anti‐human Ang‐1 (Santa Cruz, sc‐6319); and anti‐mouse Ang‐2 (R&D Systems, AF7186).

Techniques: Immunofluorescence, Staining, Control, Flow Cytometry, Comparison

Journal: Viruses

Article Title: Cytolytic Recombinant Vesicular Stomatitis Viruses Expressing STLV-1 Receptor Specifically Eliminate STLV-1 Env-Expressing Cells in an HTLV-1 Surrogate Model In Vitro

doi: 10.3390/v14040740

Figure Lengend Snippet: Amino acid sequence identity between human GLUT1 and Japanese macaque GLUT1.

Article Snippet: At 1 day post-infection, the cells were stained with mouse monoclonal anti-Human GLUT1 antibody (Ab) (R&D Systems, Minneapolis, MN, USA), followed by incubation with a FITC-conjugated goat anti-mouse IgG (H + L) Ab (Jackson ImmunoResearch, West Grove, PA, USA) in accordance with the manufacturer’s recommendations.

Techniques: Sequencing

Journal: Viruses

Article Title: Cytolytic Recombinant Vesicular Stomatitis Viruses Expressing STLV-1 Receptor Specifically Eliminate STLV-1 Env-Expressing Cells in an HTLV-1 Surrogate Model In Vitro

doi: 10.3390/v14040740

Figure Lengend Snippet: Schematic showing the structure of rVSVs expressing an STLV-1 receptor molecule and AcGFP1 as a reporter protein. The gene orders in the wildtype VSV and the rVSV constructs used in this study are illustrated. The deleted G gene was replaced with the JM GLUT1 or AcGFP1 genes, yielding the VSVΔG-JmGL or VSVΔG-AcGFP constructs, respectively. In addition, a JM GLUT1 gene was inserted upstream of the AcGFP1 gene in VSVΔG-AcGFP, yielding VSVΔG-JmGL-AcGFP.

Article Snippet: At 1 day post-infection, the cells were stained with mouse monoclonal anti-Human GLUT1 antibody (Ab) (R&D Systems, Minneapolis, MN, USA), followed by incubation with a FITC-conjugated goat anti-mouse IgG (H + L) Ab (Jackson ImmunoResearch, West Grove, PA, USA) in accordance with the manufacturer’s recommendations.

Techniques: Expressing, Construct

Journal: Viruses

Article Title: Cytolytic Recombinant Vesicular Stomatitis Viruses Expressing STLV-1 Receptor Specifically Eliminate STLV-1 Env-Expressing Cells in an HTLV-1 Surrogate Model In Vitro

doi: 10.3390/v14040740

Figure Lengend Snippet: Expression of the STLV-1 receptor molecule on the rVSV-infected target cell surface. The surface expression of JM GLUT1 protein after VSV infection was confirmed by immunofluorescence staining. VSV-permissive BHK-21 cells were infected with G-complemented VSVΔG-JmGL at an MOI of 0.1. After 1 day of culture, JM GLUT1 expressed by the viral genome was stained with anti-GLUT1-specific mouse monoclonal antibody (MAb) followed by FITC-conjugated anti-mouse IgG. The stained cells were observed by fluorescence microscopy and photographed at a constant magnification. Scale bars in panels represent 400 µm.

Article Snippet: At 1 day post-infection, the cells were stained with mouse monoclonal anti-Human GLUT1 antibody (Ab) (R&D Systems, Minneapolis, MN, USA), followed by incubation with a FITC-conjugated goat anti-mouse IgG (H + L) Ab (Jackson ImmunoResearch, West Grove, PA, USA) in accordance with the manufacturer’s recommendations.

Techniques: Expressing, Infection, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: Viruses

Article Title: Cytolytic Recombinant Vesicular Stomatitis Viruses Expressing STLV-1 Receptor Specifically Eliminate STLV-1 Env-Expressing Cells in an HTLV-1 Surrogate Model In Vitro

doi: 10.3390/v14040740

Figure Lengend Snippet: Functional assessment of infectivity and specificity of rVSVs. ( A , B ) STLV-1 Env-dependent infectivity of rVSVs was evaluated. BHK-21 cells were initially mock-transfected (empty vector) or transfected with expression plasmids for STLV-1 Env derived from Si-2 cells or from STLV-1-infected JM PBMCs. After 24 h, transfected BHK-21 cells were infected with non-G-complemented rVSVs (0.25 mL each). After 3 days of culture, the cells were fixed. Subsequently, VSVΔG-JmGL-infected cells were stained with anti-GLUT1-specific MAb to detect rVSV-infected cells. On the other hand, VSVΔG-JmGL-AcGFP- or VSVΔG-AcGFP-infected cells were detected by GFP expression. Representative results from each group of rVSV-infected BHK-21 cells are shown in panel ( A ). Areas enclosed with squares are enlarged in panel ( B ). Syncytia with enlarged cell size in VSVΔG-JmGL- or VSVΔG-JmGL-AcGFP-infected cells are shown in panel ( B ). Scale bars in panels ( A , B ) represent 400 µm and 200 µm, respectively. ( C ) STLV-1 Env-dependent rVSV infection was assessed by neutralizing assay. BHK-21 cells were initially transfected with the Si-2 strain STLV-1 Env expression plasmid. After 24 h, transfected BHK-21 cells were infected with G-complemented VSVΔG-AcGFP (MOI of 0.01) or non-G-complemented rVSVs (0.25 mL each), in the absence or presence of HTLV-1 envelope glycoprotein-specific neutralizing antibody (LAT-27; 10 µg/mL). The cells infected with VSVΔG-JmGL or VSVΔG-huGL were stained and examined by VSV N-specific immunofluorescence to detect the rVSV-infected cells at 48 h post-infection. The culture of each virus without LAT-27 was defined as a control. The total number of fluorescent cells per well was determined, and the relative infectivity of each virus was calculated. The data are expressed as a percentage of the control (mean ± SD) from four independent experiments. ( D ) Viral growth kinetic assay results are shown. Si-2 strain STLV-1 Env-transfected BHK-21 cells were infected with non-G-complemented VSVΔG-AcGFP (square) or VSVΔG-JmGL-AcGFP (circle) (0.25 mL each). The total number of GFP-positive cells per well was counted to determine the quantitative viral titer (in infectious units per milliliter). The data are expressed as the mean ± SD from four independent experiments. Asterisks in panel ( C ) represent significant differences versus control (** p < 0.001 significance, by two-tailed Student’s t -test with equal variance).

Article Snippet: At 1 day post-infection, the cells were stained with mouse monoclonal anti-Human GLUT1 antibody (Ab) (R&D Systems, Minneapolis, MN, USA), followed by incubation with a FITC-conjugated goat anti-mouse IgG (H + L) Ab (Jackson ImmunoResearch, West Grove, PA, USA) in accordance with the manufacturer’s recommendations.

Techniques: Functional Assay, Infection, Transfection, Plasmid Preparation, Expressing, Derivative Assay, Staining, Neutralizing Assay, Immunofluorescence, Virus, Control, Kinetic Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Activation-induced shift in nutrient preference and function-specific nutrient dependence in human neutrophils

doi: 10.1101/2023.09.25.559385

Figure Lengend Snippet: a. Schematic of glucose uptake, glycogen synthesis, and glycogenolysis. Important enzymes are listed. b. Glucose consumed from the media within 1 hour by unstimulated neutrophils or neutrophils that are treated with indicated stimuli (300μg/mL zymosan A, 100ng/mL TNFα, or 100nM PMA) or inhibitor (20μM GPi). Bars and error bars show mean ± SD from n=3-4 independent donors, as represented by individual dots. Statistical analysis was done by unpaired t-test, * indicates p<0.05. c. (Left) Representative flow cytometry result measuring the distribution of GLUT1 level on cell surface within a population of neutrophils that are untreated or treated with indicated stimuli or inhibitor for 30min. (Right) The experiment was repeated with neutrophils isolated from n=4 independent donors (indicated by different markers). Bars and error bars show mean ± SD of quantified mean GLUT1 level from each donor. Statistical analysis was performed by paired t-test. d-e. Immunoblot analysis of (d) total GLUT1 level and phosphorylated GLUT1 (Ser226), and (e) total PYGL and phosphorylated PYGL (Ser15) in neutrophils treated with indicated stimuli or inhibitor for 30min. Representative blots were shown on the left. The experiment was repeated multiple times with neutrophils isolated for different donors (indicated by individual dots), and quantified results were compiled and shown on the right. Bars and error bars show mean ± SD. Statistical analysis performed by unpaired t-test. f. Level of glycogen store in human peripheral blood neutrophils that are untreated, treated with GPi (20μM) for 2h, or stimulated with zymosan A (300μg/mL), or LPS (1μg/mL) for 2h. Bars and error bars show mean ± SD from n=4-5 independent donors, as represented by individual dots. p-value determined by paired t-test. g. Fraction of labeled glycogen after 2hr incubation of neutrophils that are untreated or treated with indicated stimuli or inhibitor in media containing U- 13 C-glucose. Bars and error bars show mean ± SD from n=3-5 independent donors, as represented by individual dots. p-values were determined by paired t-test.

Article Snippet: Primary antibodies: PYGL (abcam, ab198268), phospho(S15)-PYGL (abcam, ab227043), GLUT1 (Alomone Labs, AGT-041), P-GLUT1 (Millipore Sigma, ABN991), B-actin (Cell Signaling, no. 4967); and secondary antibodies: Goat-anti-rabbit 800 (LI-COR, no. 925-32211), Goat-anti-mouse 680 (LI-COR, no. 925-68070).

Techniques: Flow Cytometry, Isolation, Western Blot, Labeling, Incubation