Journal: International Journal of Cancer

Article Title: Co-expression of osteopontin and CD44v9 in gastric cancer

doi: 10.1002/(SICI)1097-0215(19980417)79:2%3C127::AID-IJC5%3E3.0.CO;2-V

Figure Lengend Snippet: FIGURE 3 – In situ hybridization for OPN mRNA in human gastric carcinomas (a–f) and immunohistochemistry of OPN (g, h) and CD44v9 (i, j) in human stomach cancers and their lymph node metastases. Positive reactions are denoted by red-color reaction product. a: well differentiated stomach cancer (case 537). c and d: tumor cells that had massive invasion into muscularis propria (case 93-4502) and lymph node metastasis. Intense staining for OPN mRNA was observed in the cytoplasm of cancer cells. b: negative control. e and f: primary tumor (case 309) and lymph node metastasis (case 93-5390). Inflammatory cells in tumor stroma showed strong positive signals for OPN mRNA (arrow heads). g and i: intralymphatic vascular component in poorly differentiated stomach cancer. Tumor cells exhibit cluster formation in lymphatic vessel and showed intense staining for both OPN and CD44. h and j: lymph node metastasis of poorly differentiated type stomach cancer (case 93-5390). Co-immunoreaction of OPN and CD44v9 was retained in metastatic focus. Scale bar, 50 µm.

Article Snippet: The sections were treated consecutively at room temperature with anti-CD44v9 mAb (diluted 1:2000) or anti-OPN antibody (diluted 1:200) for 3 hr, biotinylated anti-rabbit or mouse IgG horse serum (diluted 1:100, Vector, Burlingame, CA) for 30 min, and avidin DH-biotinylated horseradish peroxidase complex (Vectastain ABC kit, Vector) for 30 min. Peroxidase staining was performed for 10–15 min using a solution of 3,38-diaminobenzidine tetrahydrochloride in 50 mM Tris-HCl (pH 7.5) containing 0.001% hydrogen peroxide.

Techniques: In Situ Hybridization, Immunohistochemistry, Staining, Negative Control

Journal: Journal of Biological Chemistry

Article Title: SHAP Potentiates the CD44-mediated Leukocyte Adhesion to the Hyaluronan Substratum

doi: 10.1074/jbc.m506703200

Figure Lengend Snippet: FIGURE 3. Comparison between immobilized SHAPHA complex and HA on the avidities to CD44-positive Hut78 cells under flow shearing condi- tions.Theconditionsaredescribedunder“MaterialsandMethods.”Theflow- ing and rolling of cells was video-recorded, and the numbers of the firmly adheredcells(A)androllingcells(B)atvariousflowshearforceswerecounted and plotted against the shear force, respectively.

Article Snippet: Biotinylated rat IgG2a anti-mouse CD44 (clone KM81)was fromCedarlane Laboratories (Ontario, Canada).Goat anti-human TSG6 polyclonal antibody was from R&D systems, Inc. (Minneapolis, MN).

Techniques: Comparison, Shear

Journal: Journal of Biological Chemistry

Article Title: SHAP Potentiates the CD44-mediated Leukocyte Adhesion to the Hyaluronan Substratum

doi: 10.1074/jbc.m506703200

Figure Lengend Snippet: FIGURE 5. Blockage of cell-surface CD44 with anti-CD44 antibody abrogates the cell adhesion to the immobilized SHAPHA complex. A, flow cytometry analysisofHut78cellswiththreeanti-CD44antibodies.Dashedline,PBSonly;solidline,antibodytreated.Anti-humanCD44antibodyAb4andanti-mouseCD44 antibodyIM7,butnotanti-mouseCD44antibodyKM81,recognizethecellsurfaceCD44.B,pretreatmentofHut78cellswiththefirsttwoantibodiescompletely abrogated the cell adhesion, whereas neither KM81 nor the unrelated anti-II antibody showed any effect.

Article Snippet: Biotinylated rat IgG2a anti-mouse CD44 (clone KM81)was fromCedarlane Laboratories (Ontario, Canada).Goat anti-human TSG6 polyclonal antibody was from R&D systems, Inc. (Minneapolis, MN).

Techniques: Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: SHAP Potentiates the CD44-mediated Leukocyte Adhesion to the Hyaluronan Substratum

doi: 10.1074/jbc.m506703200

Figure Lengend Snippet: FIGURE 6. Reverse transcription-PCR analysis reveals the expression of CD44 standard form as well as variant forms in Hut78 cells. Liposarcoma that expresses predominantly as the standard form of CD44 was used as a control. In addition to the CD44H-derived product, two variant form-derived products were found in Hut78 cells. By the cloning and sequencing of the PCR products, it was found that the longer one was derived from CD44E (contain- ing variable exons V8, V9, and V10), whereas the shorter one was a mixture of fragments derived from two variant forms, one containing V8 and V9, and the other containing V10.

Article Snippet: Biotinylated rat IgG2a anti-mouse CD44 (clone KM81)was fromCedarlane Laboratories (Ontario, Canada).Goat anti-human TSG6 polyclonal antibody was from R&D systems, Inc. (Minneapolis, MN).

Techniques: Reverse Transcription, Expressing, Variant Assay, Control, Derivative Assay, Cloning, Sequencing

Journal: Journal of Biological Chemistry

Article Title: SHAP Potentiates the CD44-mediated Leukocyte Adhesion to the Hyaluronan Substratum

doi: 10.1074/jbc.m506703200

Figure Lengend Snippet: FIGURE 7. Activated CD44-transfected Jurkat cells adhere more firmly to the SHAPHA substratum than to the HA substratum. To the left is shown the flow cytometry analysis results of CD44-transfected Jurkat cells using Ab4 antibody. The cells were stimulated with phorbol ester to induce the HA avidity before the adhesion assay. The cells adhered more firmly to the immobilized SHAPHA complex than to the immobilized HA, a behavior resembling that of CD44-positive Hut78 cells.

Article Snippet: Biotinylated rat IgG2a anti-mouse CD44 (clone KM81)was fromCedarlane Laboratories (Ontario, Canada).Goat anti-human TSG6 polyclonal antibody was from R&D systems, Inc. (Minneapolis, MN).

Techniques: Transfection, Flow Cytometry, Cell Adhesion Assay

Journal: Journal of Biological Chemistry

Article Title: SHAP Potentiates the CD44-mediated Leukocyte Adhesion to the Hyaluronan Substratum

doi: 10.1074/jbc.m506703200

Figure Lengend Snippet: FIGURE 9. Confocal microscopic analysis of condensation of SHAPHA com- plexonthecellsurfaceofCD44-bearingcells.Hut78cellswereadheredtothe SHAPHA or HA-coated cover glass by incubating for 20 min, stained for CD44 (red)andHA(green),andexaminedwithaconfocalfluorescencemicroscope.HA was evenly distributed on the glass surface in the absence of cells. The cell surfaces were strongly positive for CD44 and HA. Section images were taken at a position 3 m above the bottom surface (A) and at the bottom surfaces (B). (The sizes of cells ranged from 12 to 16 m in diameter and 10 to 12 m in height.) Stronger signals of HA on cell surfaces were found, suggesting the condensation of HA to the cell-surface CD44, especially on the flank surfaces of cells. Note that such a condensation of HA occurs far more frequently on the surface of cells adhered to the SHAPHA substratum than to the HA sub- stratum. Almost identical patterns were observed on the samples obtained after the 1-h incubation. (Magnification: 400.)

Article Snippet: Biotinylated rat IgG2a anti-mouse CD44 (clone KM81)was fromCedarlane Laboratories (Ontario, Canada).Goat anti-human TSG6 polyclonal antibody was from R&D systems, Inc. (Minneapolis, MN).

Techniques: Staining, Incubation

Journal: Journal of Biological Chemistry

Article Title: SHAP Potentiates the CD44-mediated Leukocyte Adhesion to the Hyaluronan Substratum

doi: 10.1074/jbc.m506703200

Figure Lengend Snippet: FIGURE 11. Representative immunohistology of the hyperplastic synovium of RA patients (Steinbrock- er’s stage III). The synovium is extensively stained by HABP and anti-II antibody (counterstained with methyl green). The negative staining by anti-bikunin antibody (counterstained with hematoxylin) indicates that the immunoreactivity corresponds to the SHAPHA complex rather than II. Compared with the distribution pat- tern of HA, that of SHAP is patchier. The infiltrated CD44-positive leukocytes (counterstained with methyl green) are strongly positive on their surfaces for HA and SHAP staining. The arrow and arrowhead indicate the leukocyte and synoviocyte, respectively. Similar staining patterns were obtained with the samples at stage IV. (Magnification: 400.)

Article Snippet: Biotinylated rat IgG2a anti-mouse CD44 (clone KM81)was fromCedarlane Laboratories (Ontario, Canada).Goat anti-human TSG6 polyclonal antibody was from R&D systems, Inc. (Minneapolis, MN).

Techniques: Staining, Negative Staining

Journal: Retrovirology

Article Title: P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells

doi: 10.1186/s12977-022-00593-5

Figure Lengend Snippet: Virions circulating in vivo in HIV-infected patients contain PSGL-1 and CD44. A Plasma samples from viremic HIV-infected patients ranging from acute/early to chronic stages of HIV-1 infection were tested in virion capture assays using an isotype control antibody (IgG control), anti-PSGL-1 or anti-CD44. Captured virus was lysed, followed by RNA extraction and quantitative real-time PCR for the detection of HIV-1 genome equivalents (in RNA copies/mL). The sample median for each antibody capture condition is displayed along with results of a Mann–Whitney test (***P < 0.001). Bonferroni correction was used for adjustment of statistical significance. Each unique symbol represents a different patient, with open circles denoting patients in the acute/early stage infection and filled circles as patients in the chronic stage of infection

Article Snippet: For virion capture assays in patient plasmas, we used biotinylated antibodies, including CD44-bio (Miltenyi, Cat#130-113-340) and an in-house biotin conjugation (Bio-Rad Laboratories, Cat#LNK262B) of anti-PSGL-1 and mouse IgG1 isotype control (R&D, Cat#MAB002) antibodies.

Techniques: In Vivo, Infection, Clinical Proteomics, Control, Virus, RNA Extraction, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Potential of CD16 on Plasma-Derived Exosomes as a Liquid Biomarker in Head and Neck Cancer

doi: 10.3390/ijms21113739

Figure Lengend Snippet: CD16 levels on plasma-derived exosomes from HNSCC patients. Total exosomes or CD44v3(+) tumor-derived exosomes (TEX) isolated from plasma of HNSCC patients (n = 53 or n = 33) were stained for CD16. Surface levels as determined by on-bead flow cytometry are shown as RFI compared to an appropriate isotype control. Bars represent mean with SD. p values were determined by Mann–Whitney test, with **** corresponding to p ≤ 0.0001. Representative flow cytometry histograms depicting CD16 levels on total exosomes and CD44v3(+) TEX are shown. The solid line represents the CD16 signal, the dashed line represents the isotype control.

Article Snippet: Briefly, exosomes (10 µg in 100 µL PBS) were incubated for 2 h at RT with biotin-labeled anti-CD63 (BioLegend, San Diego, CA, USA, 353018) for total exosome capture or with biotin-labeled anti-CD44v3 (R&D Systems, Minneapolis, MN, USA, BBA11) for the enrichment of TEX.

Techniques: Derivative Assay, Isolation, Staining, Flow Cytometry, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Potential of CD16 on Plasma-Derived Exosomes as a Liquid Biomarker in Head and Neck Cancer

doi: 10.3390/ijms21113739

Figure Lengend Snippet: Correlations of CD16 levels on plasma-derived total exosomes and CD44v3(+) TEX with clinicopathological data. Significant correlations were observed between CD16 levels on total exosomes and UICC status ( A ) as well as tumor stage ( B ). No significant correlation was observed between CD16 levels on total exosomes and nodal status ( C ). No significant correlations were observed between CD16 levels on CD44v3(+) TEX and UICC status ( D ), tumor stage ( E ), or nodal status ( F ). Results are shown as RFI. Bars represent mean with SD from n = 53 ( A – C ) and n = 33 ( D – F ) HNSCC patients. p values were determined by Mann–Whitney test, with * and ** corresponding to p ≤ 0.05 and p ≤ 0.01, respectively.

Article Snippet: Briefly, exosomes (10 µg in 100 µL PBS) were incubated for 2 h at RT with biotin-labeled anti-CD63 (BioLegend, San Diego, CA, USA, 353018) for total exosome capture or with biotin-labeled anti-CD44v3 (R&D Systems, Minneapolis, MN, USA, BBA11) for the enrichment of TEX.

Techniques: Derivative Assay, MANN-WHITNEY