Journal: Frontiers in Immunology

Article Title: Low-Dose Gemcitabine Treatment Enhances Immunogenicity and Natural Killer Cell-Driven Tumor Immunity in Lung Cancer

doi: 10.3389/fimmu.2020.00331

Figure Lengend Snippet: List of primers used in qRT-PCR.

Article Snippet: Anti-human ULBP1, ULBP2/5/6, and ULBP3 antibody were purchased from R&D Systems.

Techniques:

Journal: Frontiers in Immunology

Article Title: Low-Dose Gemcitabine Treatment Enhances Immunogenicity and Natural Killer Cell-Driven Tumor Immunity in Lung Cancer

doi: 10.3389/fimmu.2020.00331

Figure Lengend Snippet: Gemcitabine up-regulates NKG2D ligands in lung cancer cells. (A) qRT-PCR of H60, Raet-1 , and Ulbp1 mRNA in LLC cells. (B) qRT-PCR of major histocompatibility complex class I polypeptide-related sequence A ( MICA ), MICB , and UL16 binding protein ( ULBP ) 1-6 mRNA in A549 cells. Data are the fold-change of mRNA expression in gemcitabine- and cisplatin-treated cells relative to untreated cells. (C) Surface expression of MICA/B, ULBP1, ULBP2/5/6, and ULBP3 was measured by flow cytometry on A549 cells treated with vehicle control (DMSO), gemcitabine or cisplatin. Data are representative of three independent experiments. One-way analysis of variance (ANOVA) was used. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Anti-human ULBP1, ULBP2/5/6, and ULBP3 antibody were purchased from R&D Systems.

Techniques: Quantitative RT-PCR, Immunopeptidomics, Sequencing, Binding Assay, Expressing, Flow Cytometry, Control

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: MDSCs identification. A, the indicated myeloid cell subsets were tested for suppressive activity against CFSE-labeled autologous T cells stimulated with beads coated with anti-CD3/anti-CD28 antibodies. Data normalized on the control (no MDSC) are cumulative of five independent experiments using PBMCs from 5 patients. P value for the ANOVA test (Pa) and the Tukey post hoc test are reported. B, example of multicolor FACS analysis for MDSC phenotype CD33+IL4Rα+ cells are highlighted in blue. C, intratumoral CD33+ IL4Rα+ cells were retrospectively evaluated in the tumor specimen of recurrent or nonrecurrent OSCC patients by immunofluorescence microscopy. P value for t test is reported.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Activity Assay, Labeling, Control, Immunofluorescence, Microscopy

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: An intermediate tadalafil dose modulates most effectively tumor immunity. The ratio between the MDSCs (A) or the log2-ratio of the CD8 proliferation (B) after (t2) and before (t1) pharmacologic treatment was plotted against the weight-normalized tadalafil dose. Best-fitting quadratic curve and confidence interval (gray area) are reported. cGMP (C) and cAMP (D) were measured by ELISA in the following FACS-sorted cell population from patients (n = 3) treated with intermediate or high dosage of tadalafil: CD33+IL4Rα+ (MDSCs), HLADRhigh (APC), or CD3+ (T cells). Pt, paired t test; BDL, below detection limit.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: Tadalafil modulates tumor microenvironment. CD33/IL4Rα (A), CD4/FoxP3 (B), or CD8/CD69 (C) intratumoral concentration was evaluated by immune-fluorescence microscopy. Pa, P ANOVA test.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Concentration Assay, Fluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: Serpina3n attenuates granzyme B-mediated decorin cleavage and rupture in a murine model of aortic aneurysm

doi: 10.1038/cddis.2011.88

Figure Lengend Snippet: GZMB cleavage of decorin is prevented by pre-incubation with SA3N in vitro . Purified GZMB was pre-incubated with or without SA3N for 25 min at RT before incubation with recombinant human decorin for 24 h at RT. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane and visualized by western blotting for decorin ( a ) and Ponceau stain ( b ). In a , full-length decorin is indicated by an asterisk ( * ) and is visible at ∼65 kDa in lane 1. Decorin is severely reduced following incubation with GZMB in lane 2. In lane 3, decorin loss is not observed following pre-incubation of GZMB with SA3N. In b , SA3N and SA3N–GZMB complex at approximately 47 and 70 kDa, respectively (indicated by ^) are visible in lanes 2, 3 and 6. Full-length decorin is indicated by the asterisk ( * ) at approximately 65 kDa in lanes 4, 5 and 6. Decorin cleavage fragments in lane 5 are marked by arrows on the right side of the membrane

Article Snippet: The membrane was probed using a mouse anti-human decorin antibody (1 : 200, R&D Systems) and IRDye 800 conjugated affinity purified anti-mouse IgG (1 : 3000, Rockland Inc., Gilbertsville, PA, USA).

Techniques: Incubation, In Vitro, Purification, Recombinant, SDS Page, Membrane, Western Blot, Staining

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) The extracellular domain of mouse VEGFR-3 was immobilized on microtiter wells and incubated with the X6 phage display library. Bar graph shows enrichment in the number of phage recovered [in transducing units (TU)] after consecutive rounds of selection (I, II, and III). (*) Round I was not quantified to prevent the loss of phage displaying unique peptides. ( B ) Peptide identified by sequencing phage bound to VEGFR-3 (round III) ( n , number of phages sequenced). ( C and D ) Binding of control phage Fd (white bars) and phage PCAIWF (B, black bars) and WVCSGG (C, black bars) to VEGF receptors and co-receptors immobilized on microtiter wells. ( E and F ) Inhibition of phage PCAIWF (E) or WVCSGG (F) binding to immobilized VEGFR-3 by synthetic peptide PCAIWF or control peptide (CARAC). The minus sign indicates that no synthetic peptide was added to the assay. ( G ) Dose-response assay. Phage PCAIWF was incubated with immobilized VEGFR-3 in the presence of synthetic peptides PCAIWF, PSAIWF, or CARAC (control). Percentage relative to phage binding in the absence of competing peptide. In all cases, bars represent means ± SEM from triplicate plating. Statistics, Student’s t test (** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Incubation, Selection, Sequencing, Binding Assay, Control, Inhibition

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Binding of phage PCAIWF to immobilized VEGFR-3 in the presence or absence of VEGF-A or VEGF-C (10 ng/ml). ( B ) Binding of phage PCAIWF to immobilized VEGFR-3 in the presence of increasing concentrations of VEGF-C. Percentage relative to phage binding in the absence of VEGF-C. ( C ) Cartoon showing the three-dimensional structure of the complex VEGF-C (red) bound to VEGFR-2 IgD2-3 (shown in orange and green, respectively) (Protein Data Bank #2X1W). ( D ) Analysis by SDS–polyacrylamide gel electrophoresis of purified recombinant IgD2 and IgD2-3 proteins containing the ligand-binding domain of VEGFR-3. ( E ) Binding of phage PCAIWF to VEGFR-3 and its recombinant Ig domains immobilized on microtiter wells in the presence or absence of the synthetic peptide PCAIWF or its scramble version, IFCAPW (100 μg/ml). Phage binding was quantified by FLISA using an anti-bacteriophage sera. ( F ) Binding of VEGF-C to microtiter wells coated with immobilized recombinant ligand binding domains IgD2 and IgD2-3 of VEGFR-3 in the presence or absence of synthetic peptides PCAIWF and WVCSGG or the scramble control peptide (IFCAPW). For phage experiments (A and B), bars represent mean ± SEM from triplicate plating; for FLISA assays ( E to G ), bars represent means ± SEM from duplicate wells. Statistics, Student’s t test [not significant (N.S.), P > 0.05; * P ≤ 0.05 and *** P ≤ 0.001].

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Binding Assay, Polyacrylamide Gel Electrophoresis, Purification, Recombinant, Ligand Binding Assay, Fluorophore-linked Immunoabsorbent Assay, Control

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Representation of the VEGF family, their receptors, and pattern of interaction. ( B to F ) Recombinant proteins for the human VEGFR-3 (B), VEGFR-2 (C and E), and VEGFR-1 (D and F) extracellular domains were immobilized on microtiter wells and incubated with the human ligands VEGF-C (B and C), PlGF (D), and VEGF-A (E and F) in the presence or absence of synthetic peptides PCAIWF and PSAIWF or the scramble control peptide (IFCAPW). Growth factors bound to the wells were quantified by FLISA using immunospecific antibodies and fluorescent detection. Bars represent means ± SEM from duplicate wells. Statistics, analysis of variance (ANOVA) (Tukey’s multiple comparison test) (* P ≤ 0.05; ** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Recombinant, Incubation, Control, Fluorophore-linked Immunoabsorbent Assay, Comparison

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Immunoblot analysis of phosphorylated and total forms of ERK1/2 in LECs incubated with VEGF-A, VEGF-C, or FGF (100 ng/ml) in the presence or absence of peptide PCAIWF or scramble (IFCAPW) (30 μg/ml). ( B ) Ratio of fluorescent intensity for phosphorylated and total ERK1/2. Bars represent means ± SEM from three independent measurements of the immunoblot membrane. Two independent experiments were performed with similar results. Bars represent means ± SEM from triplicate readings. Statistics, ANOVA (Tukey’s multiple comparison test) (* P ≤ 0.05).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Western Blot, Incubation, Membrane, Comparison

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Tube formation by HUVECs in Matrigel induced by VEGF or VEGF-C in the presence or absence of peptide PCAIWF or scramble (500 μg/ml, embedded in the Matrigel layer). ( B ) Number of tubes formed between endothelial cells. Bars represent means ± SEM from triplicate wells. Statistics, Student’s t test (* P ≤ 0.05). Two independent experiments were performed with similar results.

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: