Journal: OncoImmunology

Article Title: An epitope-specific novel anti-EMMPRIN polyclonal antibody inhibits tumor progression

doi: 10.1080/2162402x.2015.1078056

Figure Lengend Snippet: Figure 1: EMMPRIN structure and peptide design, and specificity of 161-Ab: (A) Partial

Article Snippet: One strip was probed with the 1:1,000 diluted commercial rat anti-mouse EMMPRIN (MAB772, R&D systems) and then with the 1:5,000 diluted HRP-conjugated goat anti-rat IgG (112-035- D ow nl oa de d by [ U ni ve rs ity o f R eg in a] a t 0 6: 58 2 4 Fe br ua ry 2 01 6 062, Jackson) and served as positive control (P.C).

Techniques:

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 1. Validation of the CT26-KD cells. The parental CT26 cells (WT) and knocked-down CT26 cells (KD) were seeded (8 × 104 cells each) in 24-well plates in 400 µL full medium for 48 h. At the end of the incubation, (A) total RNA was extracted from the cells and amplified using EMMPRIN specific primers (n = 4), and (B) the supernatants were collected for an ELISA analysis of EMMPRIN secretion (n = 9). Data are presented as means ± SE and analyzed using the two-tailed Student’s t test analysis. (C) CT26-WT and CT26-KD cells (30,000 cells/well/300 µL) were stained as described in the methods. A representative image, demonstrating reduced EMMPRIN protein expression in the CT26-KD cells (n = 3). Bar size is 20 µM. (D) EMMPRIN is known to appear in several bands, reflecting its low and high glycosylation patterns. Western blot analysis demonstrates that in the CT26-KD cells, all EMMPRIN bands showed a reduced expression of EMMPRIN.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Biomarker Discovery, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Staining, Expressing, Glycoproteomics, Western Blot

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 2. Co-culturing enhances the secretion of EMT-driver cytokines. CT26-WT or CT26-KD cells (80,000 cells each) were each cultured alone or co-cultured with RAW 264.7 cells that were seeded in the upper chamber of the inserts (0.4 µm pore size) at a ratio of 1:1, in serum-starvation medium (final volume 650 µL) for 48 h. At the end of the incubation, supernatants were collected and the concentrations of (A) TGFβ (n = 5), (B) soluble EMMPRIN (n = 6), (C) TNFα (n = 6), and (D) IL-6 (n = 6) were determined by ELISA. Data are presented as means ± SE, and analyzed using a two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Cell Culture, Pore Size, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 4. Expression of the EMT-TFs and dormancy markers is enhanced in the CT26-KD cells, but reduced in the co-culture or its simulation. CT26-WT or CT26-KD cells (8 × 104 cells each) were incubated alone or in co-culture with RAW 264.7 cells as described before for 48 h. Alternatively, single cultures of CT26-WT or CT26-KD cells (8 × 104 cells) were cultured with or without the addition of recombinant TGFβ (10 ng/mL) or recombinant EMMPRIN (25 ng/mL or 250 ng/mL). Total RNA was extracted from the CT26 cells, cDNA was prepared, and the genes for the dormancy markers NR2F1 and p21 or the EMT-TFs Slug and Zeb1 were amplified by qPCR as described in the methods. (A–D) cells incubated in co-cultures (n = 5–6), (E–H) single cultures incubated with the addition of TGFβ (10 ng/mL) (n = 5–6), and (I–L) cells incubated with the addition of recombinant EMMPRIN (25 and 250 ng/mL) (n = 5). Data are presented as means ± SE, and analyzed using a two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Expressing, Co-Culture Assay, Incubation, Cell Culture, Recombinant

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 5. CT26-KD exhibits reduced proliferation. CT26-WT or CT26-KD cells (8 × 104 cells) were incubated under the same conditions as described in Figure 4. Cell proliferation was measured using (A,D,G) the CCK8 kit (n = 8) or (H) the BrdU kit (n = 7) as well as the expression of (B,E,I) the cyclin D1 mRNA (n = 5) or (C,F), and the Ki67 mRNA (n = 4). (A–C) Cells incubated in co-cultures, (D,F) single cultures incubated with the addition of TGFβ (10 ng/mL), and (G–I) cells incubated with the addition of recombinant EMMPRIN (25 and 250 ng/mL). Data are presented as means ± SE, and analyzed using a two-way ANOVA followed by Bonferroni’s post-hoc test. ***, p < 0.001 relative to the CT26-WT without addition of rec. EMMPRIN; $$$, p < 0.001 relative to CT26-WT with 25 ng/ml rec. EMMPRIN; &&&, p < 0.001 relative to the CT26-WT at each concentration.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Incubation, Expressing, Recombinant, Concentration Assay

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 6. CT26-KD exhibits reduced angiogenic potential, and the co-culture reverses it. The mouse endothelial cell line bEND3 (4 × 104 cells) was cultured in full medium in 96-well plates to confluency for 24 h. A scratch was made across the monolayer, detached cells were washed away, and the remaining bEND3 cells were incubated with conditioned media (CM) derived from previous experiments for 24 h, in order to allow the migration of cells to close the gap. The CM was diluted 1:2 with full medium, to a final volume of 100 µL. Images were taken before the addition of the CM (0 h) and after 24 h of incubation with the CM (24 h). (A) Representative images of the wound assay of co-cultures. Bar size is 250 µM. (B) Quantitation of the migration of bEND3 cells cultured with CM from co-culture experiments (n = 12), with (C) concentrations of VEGF (n = 7) and (D) MMP-9 (n = 7) in the supernatants derived from co-culture experiments. (E) Quantitation of the migration using CM derived from the TGFβ experiments (n = 5), and concentrations of (F) VEGF (n = 8) and (G) MMP-9 (n = 5) in the supernatants derived from TGFβ experiments. (H) Quantitation of the migration using CM derived from the recombinant EMMPRIN experiments (n = 9), and concentrations of (I) VEGF (n = 9) and (J) MMP-9 (n = 7) in the supernatants derived from recombinant EMMPRIN experiments. Data are presented as means ± SE, and analyzed using two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Co-Culture Assay, Cell Culture, Incubation, Derivative Assay, Migration, Quantitation Assay, Recombinant

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 7. The combination of recombinant TGFβ and EMMPRIN has no effect on the addition of TGFβ or EMMPRIN alone. CT26-WT or CT26-KD cells (2.5 × 104 cells) were incubated in triplicates for 48 h in serum starvation medium, with or without the addition of recombinant TGFβ (5 ng/mL), EMMPRIN (5 ng/mL), or their combination. The effect on the (A) proliferation, as measured by CCK8 (n = 10), (B) the concentrations of secreted VEGF (n = 5), as well as the mRNA expression of (C) NR2F1 (n = 6), (D) p21 (n = 6), (E) Slug (n = 6), and (F) Zeb1 (n = 6). Data are presented as means ± SE, and analyzed using a two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Recombinant, Incubation, Expressing

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

Journal: bioRxiv

Article Title: EMMPRIN confers metabolic advantage for monocytes and macrophages to promote disease in a model of multiple sclerosis

doi: 10.1101/2024.08.11.607460

Figure Lengend Snippet: A) Confocal images of perivascular cuffs in the cerebellar white matter of CCR2:EMMP +/+ and CCR2:EMMP −/− D18 EAE showing pan-leukocytic marker, CD45 (red) encased in basement membrane delineated by laminin staining (green). Insets showing enlarged cuffs with CD45+ cells within. Scale-50μm. B) Histograms denoting average number of cuffs observed in the two groups. Data analyzed by student’s T-test. **p<0.05, N of 6 per group. C) Spinal cords were harvested from CCR2:EMMP +/+ and CCR2:EMMP −/− mice at D18 after immunization and subjected to flow cytometry: Singlet viable cells were gated on CD11b and CD45 for CD45hi CD11b+ monocyte/macrophages as shown in dot-plot for CCR2:EMMP −/− and CCR2:EMMP +/+ examples; there were few monocyte/macrophages in the spinal cord of CCR2:EMMP −/− mice. D) Analysis of % EMMPRIN+ CCR2+ infiltrated macrophages as well as E) expression of EMMPRIN levels (MFI = mean fluorescence intensity) in CCR2+ CD11b+LY6G-cells. F) Dot plot exhibiting Ly6G and CD11b staining from blood of CCR2:EMMP +/+ and CCR2:EMMP −/− D12 EAE mice which is quantified as %CD11b+ Ly6G-CD45+ cells in (G) . Histograms showing %CD11b+ Ly6G-CD45+ cells from D18 EAE in WT, CCR2:EMMP +/+ and CCR2:EMMP −/− mice. Flow plots shown in D, E, G and H were compared using one-way ANOVA with Bonferroni post-hoc test. *p<0.05, **p<0.01 and ***p<0.001. Data represented as mean ± SD.

Article Snippet: Cells were then washed and incubated in following antibodies for 30 minutes at 4°C in the dark: PerCP rat anti-mouse CD45 (557235, BD Biosciences, 1:50), FITC rat anti-CD11b (553310, BD Biosciences, 1:50), APC-Cy7 rat anti-mouse Ly6G (560600, BD Biosciences, 1:50), PE rat anti-mouse EMMPRIN (562676, BD Biosciences, 1:50), BUV395 rat anti-mouse CD115 (564059, BD Biosciences, 1:50), BV510 rat anti-mouse CCR2 (CD192, 747970, BD Biosciences, 1:50) and fixable viability dye eFluor 780 (65-0865-14, eBioscience).

Techniques: Marker, Membrane, Staining, Flow Cytometry, Expressing, Fluorescence