Journal: PLoS ONE

Article Title: Antitumor and Angiostatic Activities of the Antimicrobial Peptide Dermaseptin B2

doi: 10.1371/journal.pone.0044351

Figure Lengend Snippet: PC3 cells were treated with 2.5 µM of Drs B2 during 1 hour where after fixed and stained with anti-Drs B2 polyclonal purified rabbit IgG. Drs B2 is visualized by using Fluo 546-donkey rabbit antibody (red), membranes by CD147 labeled with FITC-AffiniPure Goat Mouse antibody (green) and nuclei by DAPI (blue). Confocal fluorescence images were acquired using an IX81 inverted Olympus microscope (60× oil-immersion NA 1.25 objective) equipped with a DSU spinning disk confocal system (Olympus; Rungis, France), coupled to an Orca R2 CCD camera (Hamamatsu Corporation; Japan). Images were arranged using the image processing software ImageJ, and the program Zoom in Images and Stack. Panels 1A–C present secondary antibody controls 1A) for the Drs B2 detection by fluo 546-donkey rabbit antibody and 1B) for the CD147 detection by FITC-AffiniPure Goat Mouse antibody. Panel 1C corresponds to the composite image of 1A and 1B plus the DAPI labeling of the same field. Panels 1D–F show labeling controls for 1D) the Drs B2 detection by anti Drs B2 and fluo 546-donkey rabbit antibodies in absence of Drs B2 treatment, and for 1E) the CD147 detection by anti CD147 and FITC-AffiniPure Goat Mouse antibodies. Panel 1F corresponds to the composite image of 1D and 1E plus the DAPI labeling of the same field. Panels 2A, 3A, 4A, 5A and 6A present the maximum projection image of the composite RGB stacks along the z axis. Panels 2B–D, 3B–D, 4B–D, 5B–D and 6 B–C show the confocal image series of the same region of interest wherein z represent the distance from the base of the cell in µm (insets zoom factor = 2). Panels 2E, 3E, 4E, 5E, 6D present the 3D volume rendering of the cells from the composite RGB stacks prepared by using the software FreeSFP. Scale bar: 10 µm.

Article Snippet: Purified mouse anti-human EMMPRIN/CD147 was from BD Pharmingen (Bedford, UK) and goat anti-mouse FITC-AffiniPure was from Jackson ImmunoResearch Laboratories Inc. (West Grove, USA).

Techniques: Staining, Purification, Labeling, Fluorescence, Microscopy, Software

Journal: BMC Cancer

Article Title: EMMPRIN/CD147 up-regulates urokinase-type plasminogen activator: implications in oral tumor progression

doi: 10.1186/1471-2407-12-115

Figure Lengend Snippet: EMMPRIN and uPA expression in human oral dysplastic and squamous carcinoma lesions . A- EMMPRIN and uPA mRNA expression in oral dysplastic and tumor tissues. RNA was extracted from formalin-fixed paraffin-embedded tissue sections of 6 normal oral mucosa (Normal) , 4 intra epithelial neoplasia ( Dysplastic ), 8 micro-invasive OSCC and 8 invasive OSCC, and were analysed for EMMPRIN and uPA expression using qRT-PCR assays. * denotes significant difference with p < 0.05. B- Immunohistochemical staining of EMMPRIN and uPA in sections of human oral dysplastic, micro-invasive and invasive squamous carcinoma lesions. Tissue sections from patients with oral dysplastic lesions (OIN), micro-invasive lesions (Micro-inv) and invasive oral squamous carcinoma lesions (INV) lesion sections were subjected to single-labeled immunohistochemistry using mouse anti-human EMMPRIN mAb or goat anti-human uPA Ab and counterstained with HES. EMMPRIN and uPA were colocalized, their staining was heterogeneous with particularly high staining at some regions. Staining was more intense in the tumor sections.

Article Snippet: For immunohistochemistry, FFPE sections were stained with a mouse anti-human EMMPRIN antibody (BD-Pharmingen) and a goat anti human uPA antibody (American diagnostica).

Techniques: Expressing, Formalin-fixed Paraffin-Embedded, Quantitative RT-PCR, Immunohistochemical staining, Staining, Labeling, Immunohistochemistry

Journal: BMC Cancer

Article Title: EMMPRIN/CD147 up-regulates urokinase-type plasminogen activator: implications in oral tumor progression

doi: 10.1186/1471-2407-12-115

Figure Lengend Snippet: EMMPRIN regulates uPA in oral dysplastic DOK and tumor SCC-9 cell lines . A, (a) CHO cells were transfected with EMMPRIN cDNA as previously described . Membranes were isolated from CHO-Emp and CHO control cells (CHO-Emp Mb and CHO Mb, respectively) by differential centrifugation and 10 μg of membrane extract were analyzed for EMMPRIN content by immunoblotting. (b) DOK and SCC-9 cells (80% confluence) were incubated with 20 μg/mL CHO Mb or CHO-Emp Mb in serum-free medium for 24 h and the conditioned medium was analyzed for uPA activity using casein-plasminogen zymography and for gelatinase activities of MMP-2 and MMP-9 using gelatin zymography (one representative zymography of three independent experiments). (c) EMMPRIN, uPA, MMP-2, MMP-9 and TIMP-1 transcripts were quantified using quantitative RT-PCR in DOK and SCC-9 cells incubated for 24 h with CHO Mb or CHO-Emp Mb (20 μg/mL). Columns are means of gene expression relative to TBP housekeeping gene of at least three independent experiments carried out in triplicate; bars , SD. * denotes significant difference with p < 0.05. (d) SCC-9 and DOK cells were grown in microscope slide chambers to 50-60% confluence and incubated with CHO Mb or CHO-Emp Mb (20 μg/mL). After 24 h, cells were immunostained for uPA using a goat anti-human antibody (green) and counterstained with DAPI (blue). A more intense staining was observed in CHO-Emp mb treated DOK and SCC-9 cells compared to the control CHO mb treated cells. B , SCC-9 and DOK cells were incubated with 20 μg/mL of an anti-EMMPRIN blocking antibody (Ancell, Bayport, MN) or with a non-immune IgG antibody in serum-free medium. After 24 h incubation, conditioned medium was harvested for gelatinase and uPA casein zymography. Columns represent average values of the densitometric quantification from at least three independent experiments carried out in triplicate; bars , SD . * denotes significant difference with p < 0.05. C , DOK and SCC-9 cells were transfected with EMMPRIN siRNA (Emp-siRNA) or scrambled control siRNA (Ctl siRNA) at 33 nmol/L concentration. EMMPRIN and uPA mRNA expression was evaluated by quantitative RT-PCR analyses. Columns represent mean ± SD of relative expression to TBP housekeeping gene of at least 3 independent experiments carried out in triplicate; bars , SD. * denotes significant difference with p < 0.05.

Article Snippet: For immunohistochemistry, FFPE sections were stained with a mouse anti-human EMMPRIN antibody (BD-Pharmingen) and a goat anti human uPA antibody (American diagnostica).

Techniques: Transfection, Isolation, Centrifugation, Western Blot, Incubation, Activity Assay, Zymography, Quantitative RT-PCR, Expressing, Microscopy, Staining, Blocking Assay, Concentration Assay

Journal: BMC Cancer

Article Title: EMMPRIN/CD147 up-regulates urokinase-type plasminogen activator: implications in oral tumor progression

doi: 10.1186/1471-2407-12-115

Figure Lengend Snippet: EMMPRIN regulates invasion in oral dysplastic DOK and tumor SCC-9 cell lines in an MMP and uPA dependent manner. A , The in vitro invasive property of DOK and SCC-9 cells incubated with CHO-Emp membranes and treated or not with an anti-EMMPRIN blocking antibody (20 μg/mL) were compared using tissue culture Transwell inserts (8-mm pore size; BD Biosciences) placed in a 24-well culture plate. Cells incubated with CHO control or treated with an anti-IgG antibody were used as control. Cells (1 × 10 5 ) suspended in serum-free media were seeded into the upper well of each insert onto membranes coated with growth factor-reduced Matrigel (BD Biosciences). After 48 h incubation, cells that remained in the top compartment were removed by cotton swabs, and cells on the underside of insert filters were fixed, stained, and counted under a microscope. The columns shown represent average values from at least three independent experiments carried out in triplicate; bars , SD. * denotes significant difference with p < 0.05. B , DOK and SCC-9 cells were transfected with EMMPRIN siRNA or scrambled siRNA (Ctl siRNA) prior to invasion assays. The columns represent means of three independent experiments carried out in triplicate; bars , SD. C , Invasive Indices. DOK and SCC-9 cells (1 × 10 5 ) seeded into the upper well of tissue culture Transwell inserts coated with growth factor-reduced Matrigel (for invasion assay) or not coated (for migration assay) were incubated with CHO or CHO-Emp membranes (Control and CHO Emp mb respectively). After 48 h of incubation, migrating or invading cells on the underside of insert filters were fixed, stained, and counted under a microscope. Invasion Indices were calculated as percent of the invaded cells relative to the migrated cells. The columns shown represent average values from at least three independent experiments carried out in triplicate; bars , SD. * denotes significant difference with p < 0.05. D , Relative contribution of uPA and MMPs to the in vitro invasive property of DOK and SCC-9 cells. Cells (1 × 10 5 ) were seeded into the upper well of matrigel coated inserts and incubated with CHO or CHO-Emp membranes. uPA inhibitor amiloride (20 nmol/L) or MMP inhibitor marimastat (10 μmol/L) were added alone or in combination together with the membranes. After 48 h invading cells were fixed, stained, and counted. Columns represent average values from at least three independent experiments carried out in triplicate; bars , SD. * denotes significant difference with p < 0.05.

Article Snippet: For immunohistochemistry, FFPE sections were stained with a mouse anti-human EMMPRIN antibody (BD-Pharmingen) and a goat anti human uPA antibody (American diagnostica).

Techniques: In Vitro, Incubation, Blocking Assay, Staining, Microscopy, Transfection, Invasion Assay, Migration

Journal: Journal of Extracellular Vesicles

Article Title: Functional and transcriptomic analysis of extracellular vesicles identifies calprotectin as a new prognostic marker in peripheral arterial disease (PAD)

doi: 10.1080/20013078.2020.1729646

Figure Lengend Snippet: Characterization of isolated EVs . (a) Representative size distribution histogram for platelet-free plasma derived EVs. The NTA analysis shows a polydisperse heterogeneous vesicle population, the vast majority of them ranging from 100 to 400 nm. (b) Western blot for EVs (Alix and EMMPRIN) and non-EVs markers (ApoB100 and ApoA1) on EVs samples isolated from platelet-free plasma (n = 2). The first line on the left corresponds to the molecular weight marker (MW, kDa) of each detected protein. (c) Representative cryo-electron micrographs of EVs isolated from platelet-free plasma. Panels II and IV correspond to the insets drawn in panels I and II, respectively. EVs are round shaped and delimited by a lipid bilayer. Moreover, smaller electron dense particles (~25 nm) lacking a visible lipid membrane are observed (black arrows), indicating the presence of contaminants such as VLDL or LDL. Scale bar denotes 100 nm. (d) Representative flow cytometry dot-plot for unstained medium/large size EVs isolated from platelet-free plasma within the working gate (in blue). The gate was defined using the violet side scatter (Violet-SSC) against the regular SSC, using calibrated beads of sizes ranging from 0.25 to 1.34 µm as explained in ). (e) Representative dot-plots for gated EVs confronting the fluorescence intensity for FITC vs. APC. Processing of CFSE in EVs gives a positive signal in the FITC channel. Left panel shows no fluorescent signal in unstained EVs, while 80% of CFSE stained EVs are FITC positive (right panel).

Article Snippet: Blots were incubated overnight with primary antibodies: Alix (mouse Anti-AIP1, clone 49/AIP1, 0.125 μg/mL BD Bioscience), EMMPRIN (mouse monoclonal anti-human EMMPRIN/CD147, clone IT10C5, 1 μg/mL, Immunotools), ApoA1 (rabbit polyclonal anti-Apolipoprotein A1, sc-30089, 2 μg/mL, Santa Cruz), ApoB100 (goat polyclonal anti human Apolipoprotein B100, AF3260, 1 μg/mL, Novus Biologicals) and S100A9 (rabbit polyclonal anti-human S100A9, 0.4 μg/mL, Invitrogen) followed by 1 h incubation with required peroxidase-conjugated secondary antibodies.

Techniques: Isolation, Clinical Proteomics, Derivative Assay, Western Blot, Molecular Weight, Marker, Membrane, Flow Cytometry, Fluorescence, Staining

Journal: Cancers

Article Title: CD147 Promotes Cell Small Extracellular Vesicles Release during Colon Cancer Stem Cells Differentiation and Triggers Cellular Changes in Recipient Cells

doi: 10.3390/cancers12020260

Figure Lengend Snippet: Sequence of primer used for RT-qPCR.

Article Snippet: Primary monoclonal antibodies were used following suppliers’ instructions and included the following: mouse anti-human monoclonal CD9 (dilution, 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), mouse monoclonal anti-human EMMPRIN (dilution, 1:500; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-human EMMPRIN (8D6; sc-21746; dilution 1:500; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-human β-Actin (C4; sc-47778; dilution 1:500; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-human PARP-1 (N-20; sc-1561; dilution 1:500; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-human PROM1 (PAB12663; dilution 1:500 Abnova, Heidelberg, Germany).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans

doi: 10.1371/journal.pone.0182926

Figure Lengend Snippet: PC3 cells were treated with 2.5 μM [Alexa594]—(Cys 0 ) -DRS-B2 (red) for either 5 min. (1A-C) or 1 hour (2A-D). After fixation, the membrane was visualized by the anti-CD147 antibody labeled with an anti-mouse antibody A488 (green) and nuclei by DAPI (blue). The confocal images were obtained using an Olympus IX81 inverted microscope (60x oil immersion objective NA 1.25) with a spinning disk confocal system DSU (Olympus), coupled to a CCD camera R2 Orca (Hamamatsu Corporation, Japan). Image processing was performed using the ImageJ software, and the program Zoom in Images and Stack. z is the distance from the base to the top of the cell in 0.5 μm steps. The boxes are zoom lenses, the zoom factor = 2.

Article Snippet: Purified mouse anti-human EMMPRIN/CD147 antibodies was from BD Pharmingen (Bedford, UK)

Techniques: Labeling, Inverted Microscopy, Software

Journal: PLoS ONE

Article Title: Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans

doi: 10.1371/journal.pone.0182926

Figure Lengend Snippet: U87MG cells were treated with 2.5 μM of [Alexa594]—(Cys 0 ) -DRS-B2 (red) for either 5 min. (1A-D) or 1 hour (2A-C). After fixation, the membrane was visualized using anti CD147 antibody labeled with an anti-mouse antibody A488 (green) and nuclei by DAPI (blue). The confocal images were acquired using Olympus IX81 inverted microscope (60x oil immersion objective NA 1.25) with a spinning disk confocal system DSU (Olympus), coupled to a CCD camera R2 Orca (Hamamatsu Corporation, Japan). Image processing was performed using the program Zoom in Images and Stack. z is the distance from the base to the top of the cell in 0.5 μm steps. The boxes are zoom lenses, the zoom factor = 2.

Article Snippet: Purified mouse anti-human EMMPRIN/CD147 antibodies was from BD Pharmingen (Bedford, UK)

Techniques: Labeling, Inverted Microscopy