Journal: Blood

Article Title: Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates vesicle uptake by macrophages.

doi: 10.1182/blood-2009-07-231449

Figure Lengend Snippet: Figure 1. Galectin-5 is present on the surface of rat red cells. (A) Freshly isolated reticulocytes or erythrocytes were adsorbed on glass coverslips and processed for immunofluorescence as described in “Fluorescence-activated cell-sorting analysis of exosomes and red cells, fluorescence microscopy of red cells.” Transmission images of red cells (left) and corresponding fluorescence imaging (right) were recorded on cells by the use of purified rabbit anti–galectin-5 antibody followed by incubation withAlexa 488 anti–rabbit antibody. (B) Young reticulocytes and erythrocytes were analyzed by flow cytometry by the use of antibodies raised against Gal-2 (dashed line), Gal-4 (dotted line), and Gal-5 (solid line), already tested for their specificity (top), or the produced anti–galectin-5 serum (solid line) and the preimmune serum (bottom, dashed line). Tinted patterns indicate cell labeling obtained in the absence of primary antibodies. (C) Lymphocytes isolated from rat blood, as described in “Cells,” were analyzed by flow cytometry for Gal-5 (left, solid line), CD47 (middle, solid line) and Syto 16 green (right, solid line). Tinted patterns indicate cell labeling in the absence of primary antibodies. (D) Ghost and raft extracts isolated from reticulocytes or mature erythrocytes, as described in “Red cell subcellular fractionation,” were processed by SDS-PAGE and analyzed by Western blot for the indicated proteins. The molecular mass (kDa) standards are indicated on the left.

Article Snippet: Mouse anti–rat CD47 was from Serotec Limited.

Techniques: Isolation, Fluorescence, FACS, Microscopy, Transmission Assay, Imaging, Incubation, Cytometry, Produced, Labeling, Fractionation, SDS Page, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Targeting cluster of differentiation 47 improves the efficacy of anti-cytotoxic T-lymphocyte associated protein 4 treatment via antigen presentation enhancement in pancreatic ductal adenocarcinoma

doi: 10.3892/etm.2020.9054

Figure Lengend Snippet: Antibodies used in the flow cytometry experiments of the present study.

Article Snippet: When 70% confluency was reached, the 293 cells were transfected using Lipofectamine 3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) with aCD47 overexpression vector (cat. no. MR204706L1; OriGene Technologies, Inc.), CD47 shRNA (cat. no. TL501123; OriGene Technologies, Inc.) or control vector (cat. no. PS100064; OriGene Technologies, Inc.; 20 µg each) together with the packaging vector (Lenti-vpak packaging kit; cat. no. TR30037; OriGene Technologies, Inc.) in order to produce lentiviruses to infect Panc02 cells.

Techniques: Flow Cytometry

Journal: Experimental and Therapeutic Medicine

Article Title: Targeting cluster of differentiation 47 improves the efficacy of anti-cytotoxic T-lymphocyte associated protein 4 treatment via antigen presentation enhancement in pancreatic ductal adenocarcinoma

doi: 10.3892/etm.2020.9054

Figure Lengend Snippet: Expression of CD47 and activation of antigen-presenting cells in PDAC samples. (A) CD47 expression in normal pancreatic and paired tumor tissues was measured by fluorescence-activated cell sorting analysis (sample size=20). Tumor tissues exhibited higher CD47 expression as compared with that in normal tissues. (B) Quantified data showing that the MFI of CD47 in tumor tissues was higher than that in normal tissues. (C) DCs and (D) macrophages exhibited higher CD80 expression in human PDAC samples with low CD47 expression. (E) DCs and (F) macrophages exhibited higher CD86 expression in human PDAC samples with low CD47 expression. *P<0.05, **P<0.01 and ****P<0.0001. CD47, cluster of differentiation 47; PDAC, pancreatic ductal adenocarcinoma; MFI, mean fluorescence intensity; DCs, dendritic cells.

Article Snippet: When 70% confluency was reached, the 293 cells were transfected using Lipofectamine 3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) with aCD47 overexpression vector (cat. no. MR204706L1; OriGene Technologies, Inc.), CD47 shRNA (cat. no. TL501123; OriGene Technologies, Inc.) or control vector (cat. no. PS100064; OriGene Technologies, Inc.; 20 µg each) together with the packaging vector (Lenti-vpak packaging kit; cat. no. TR30037; OriGene Technologies, Inc.) in order to produce lentiviruses to infect Panc02 cells.

Techniques: Expressing, Activation Assay, Fluorescence, FACS

Journal: Experimental and Therapeutic Medicine

Article Title: Targeting cluster of differentiation 47 improves the efficacy of anti-cytotoxic T-lymphocyte associated protein 4 treatment via antigen presentation enhancement in pancreatic ductal adenocarcinoma

doi: 10.3892/etm.2020.9054

Figure Lengend Snippet: Effects of increased CD47 expression on phagocytosis of macrophages and DCs. (A) FACS analysis confirmed the CD47 expression manipulation in Panc02 cells (B) Representative dot plots indicating how the phagocytic index was measured by FACS method (Q3 indicates the Panc02 cells that were phagocytosed by macrophages). (C) Panc02 cells with various expression levels of CD47 were co-cultured with macrophages, and the phagocytic index was measured. Quantified data revealed that macrophages had a higher phagocytic index in CD47-KD Panc02 cells. (D) Anti-CD47 treatment significantly rescued the phagocytic function of macrophages. (E) CD47-overexpressing tumor cells inhibited the phagocytic function of DCs. (F) Anti-CD47 treatment significantly rescued the phagocytic function of DCs. All the experiments were repeated three times.**P<0.01, ***P<0.001 ****P<0.0001. CD47, cluster of differentiation 47; OE, overexpression; WT, wide type; Ctrl, control; KD, knockdown; FACS, fluorescence-activated cell sorting.

Article Snippet: When 70% confluency was reached, the 293 cells were transfected using Lipofectamine 3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) with aCD47 overexpression vector (cat. no. MR204706L1; OriGene Technologies, Inc.), CD47 shRNA (cat. no. TL501123; OriGene Technologies, Inc.) or control vector (cat. no. PS100064; OriGene Technologies, Inc.; 20 µg each) together with the packaging vector (Lenti-vpak packaging kit; cat. no. TR30037; OriGene Technologies, Inc.) in order to produce lentiviruses to infect Panc02 cells.

Techniques: Expressing, Cell Culture, Over Expression, Fluorescence, FACS

Journal: Experimental and Therapeutic Medicine

Article Title: Targeting cluster of differentiation 47 improves the efficacy of anti-cytotoxic T-lymphocyte associated protein 4 treatment via antigen presentation enhancement in pancreatic ductal adenocarcinoma

doi: 10.3892/etm.2020.9054

Figure Lengend Snippet: CD47 overexpression inhibited antigen-presenting cell infiltration and activity in a pancreatic ductal adenocarcinoma mouse model. The mouse model was established using Panc02 cell lines with various CD47 expression levels, and the ratio of macrophages and DCs was measured by the fluorescence-activated cell sorting method. (A) The representative flow plots showed gating of macrophages in tumors. The CD47-OE tumor tissues exhibited the lowest percentage of macrophages. (B) CD80 and (C) CD86 expressed in macrophages isolated from CD47-OE tumor tissues were the lowest compared with other groups. Representative histograms of signal intensity were shown for each group. (D) The representative flow plots showed gating of DCs in tumors. The CD47-OE tumor tissues exhibited the lowest percentage of DCs. (E) CD80 and (F) CD86 expression levels were significantly reduced in the DCs isolated from CD47-OE tumor tissues. Representative histograms of signal intensity were shown for each group. ***P<0.001, ****P<0.0001. CD47, cluster of differentiation 47; MFI, mean fluorescence intensity; OE, overexpression; WT, wide type; Ctrl, control; KD, knockdown.

Article Snippet: When 70% confluency was reached, the 293 cells were transfected using Lipofectamine 3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) with aCD47 overexpression vector (cat. no. MR204706L1; OriGene Technologies, Inc.), CD47 shRNA (cat. no. TL501123; OriGene Technologies, Inc.) or control vector (cat. no. PS100064; OriGene Technologies, Inc.; 20 µg each) together with the packaging vector (Lenti-vpak packaging kit; cat. no. TR30037; OriGene Technologies, Inc.) in order to produce lentiviruses to infect Panc02 cells.

Techniques: Over Expression, Activity Assay, Expressing, Fluorescence, FACS, Isolation

Journal: Experimental and Therapeutic Medicine

Article Title: Targeting cluster of differentiation 47 improves the efficacy of anti-cytotoxic T-lymphocyte associated protein 4 treatment via antigen presentation enhancement in pancreatic ductal adenocarcinoma

doi: 10.3892/etm.2020.9054

Figure Lengend Snippet: Effects of CD47 overexpression on PDAC development. A PDAC syngeneic mouse model was established using Panc02 cell lines with various CD47 expression levels. (A) Growth of tumors established by CD47-OE cells was the fastest as compared with the other groups. (B) Mice with CD47-OE tumors had the lowest survival rate as compared with the other groups (n=10 in each group). **P<0.01, ****P<0.0001 vs. CD47-WT. CD47, cluster of differentiation 47; PDAC, pancreatic ductal adenocarcinoma; OE, overexpression; WT, wide type; Ctrl, control; KD, knockdown.

Article Snippet: When 70% confluency was reached, the 293 cells were transfected using Lipofectamine 3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) with aCD47 overexpression vector (cat. no. MR204706L1; OriGene Technologies, Inc.), CD47 shRNA (cat. no. TL501123; OriGene Technologies, Inc.) or control vector (cat. no. PS100064; OriGene Technologies, Inc.; 20 µg each) together with the packaging vector (Lenti-vpak packaging kit; cat. no. TR30037; OriGene Technologies, Inc.) in order to produce lentiviruses to infect Panc02 cells.

Techniques: Over Expression, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Targeting cluster of differentiation 47 improves the efficacy of anti-cytotoxic T-lymphocyte associated protein 4 treatment via antigen presentation enhancement in pancreatic ductal adenocarcinoma

doi: 10.3892/etm.2020.9054

Figure Lengend Snippet: Anti-CD47 treatment enhanced the efficacy of anti-CTLA4 treatment via stimulating anti-tumor immunity. (A) The experimental scheme. (B) Growth of tumors co-treated with anti-CD47 and anti-CTLA4 antibodies was slower in comparison with tumors treated with monotherapy. ****P<0.0001 vs. IgG group; #P<0.05 vs. anti-CTLA4 group) (C) Mice co-treated with anti-CD47 and anti-CTLA4 antibodies exhibited longer survival as compared with mice receiving monotherapy. (D) Number of gp70+ CD8 T-cells and (E) Ki-67 expression in the tumor-draining lymph node were increased by anti-CD47 and anti-CTLA4 combination treatment. (F) Number of gp70+ CD8 T-cells and (G) Ki-67 expression levels in tumor tissues were higher in the anti-CD47 and anti-CTLA4 combination treatment group. Sample size=10 per group. *P<0.01, ***P<0.001 and ****P<0.0001. CTLA4, cytotoxic T-lymphocyte associated protein 4. CD47, cluster of differentiation 47; MFI, mean fluorescence intensity.

Article Snippet: When 70% confluency was reached, the 293 cells were transfected using Lipofectamine 3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) with aCD47 overexpression vector (cat. no. MR204706L1; OriGene Technologies, Inc.), CD47 shRNA (cat. no. TL501123; OriGene Technologies, Inc.) or control vector (cat. no. PS100064; OriGene Technologies, Inc.; 20 µg each) together with the packaging vector (Lenti-vpak packaging kit; cat. no. TR30037; OriGene Technologies, Inc.) in order to produce lentiviruses to infect Panc02 cells.

Techniques: Expressing, Fluorescence

Journal: Oncoimmunology

Article Title: IRE1α overexpression in malignant cells limits tumor progression by inducing an anti-cancer immune response

doi: 10.1080/2162402X.2022.2116844

Figure Lengend Snippet:

Article Snippet: PE-Vio770 anti-CD47 , REAfinity, Miltenyi Biotec , 130–103-105, RRID:AB_2659751.

Techniques:

Journal: Nature Communications

Article Title: Microenvironment-confined kinetic elucidation and implementation of a DNA nano-phage with a shielded internal computing layer

doi: 10.1038/s41467-025-56219-9

Figure Lengend Snippet: a The structure of a T4 bacteriophage and the structure of DNPs with different aptamer-based specific recognition toes. b The working principle of different computing layers of a DNP including the input computing layer, the shielded internal computing layer and the output computing layer. c The schematic illustration of the stoichiometric and site-specific Nb labeling strategy. d Binding competition between Nb-S and monoclonal antibody B6H12 on target cell surface. Data are represented as mean ± s.d. ( n = 3 from three independent experiments). e AFM imaging of cage-Nb. Scale bar: 50 nm. The experiment was repeated independently for 3 times with similar results. f Flow cytometry analysis and confocal imaging of CEM cells bound by cage-Nb and Nb-S to characterize the protection of DNA cage on Nb binding. Scale bar: 10 μm. Source data are provided as a Source Data file.

Article Snippet: When using monoclonal antibody B6H12-PE (cat.no.12283-MM07-P, Sino Biological, China) to complete the binding between CD47 Nb and target cell, cells were first washed with DPBS three times and incubated with Nb or Nb-S at 4 °C for 30 min. Then cells were washed with DPBS three times and incubated with B6H12-PE at 4 °C for 30 min.

Techniques: Labeling, Binding Assay, Imaging, Flow Cytometry

Journal: Nature Communications

Article Title: Microenvironment-confined kinetic elucidation and implementation of a DNA nano-phage with a shielded internal computing layer

doi: 10.1038/s41467-025-56219-9

Figure Lengend Snippet: a Flow cytometry analysis of the segregation effect of DNP3 in shielding the interference of erythrocyte. From left to right: control, Nb-S, DNP1, DNP3. The yellow dots indicate the CEM cells pre-stained with Mitochondrial Red and the gray dots indicate erythrocytes. b Hemolysis test result of Triton X-100, monoclonal antibodies B6H12 and DNP3. Data are represented as mean ± s.d. ( n = 3 from three independent experiments). c Flow cytometry result of DNP enhanced phagocytosis of CEM cells by macrophages. Boxed region in the plots indicated the percentage of macrophage that phagocytized cancer cells. d Flow cytometry-based statistical analysis of phagocytosis of different cells by macrophages. Data are represented as mean ± s.d. ( n = 2 from two independent experiments). e Phagocytosis of Far-red-labeled CEM (red) by CFSE-labeled M1 macrophages (green) after treated by Nb-S, cage-Nb, DNP3. White arrows indicate macrophages that phagocytized cancer cells. Scale bar: 50 μm. f Confocal imaging-based statistical analysis of phagocytosis of different cells by macrophages. Data are represented as mean ± s.d. ( n = 5 from five independent experiments). g Time-depended confocal imaging of DNP3-mediated phagocytosis. Macrophages were stained by CFSE (green) and CEM were labeled with Far-red (red). Source data are provided as a Source Data file.

Article Snippet: When using monoclonal antibody B6H12-PE (cat.no.12283-MM07-P, Sino Biological, China) to complete the binding between CD47 Nb and target cell, cells were first washed with DPBS three times and incubated with Nb or Nb-S at 4 °C for 30 min. Then cells were washed with DPBS three times and incubated with B6H12-PE at 4 °C for 30 min.

Techniques: Flow Cytometry, Control, Staining, Labeling, Imaging

Journal: JCI Insight

Article Title: CD47 prevents the elimination of diseased fibroblasts in scleroderma

doi: 10.1172/jci.insight.140458

Figure Lengend Snippet: ( A ) ATAC-Seq analysis for JUN and the hedgehog genes Gli1 and Ptch1 in scleroderma fibroblasts (SSCL), scleroderma fibroblasts after JUN knockout (JUN-KO) or under vismodegib, and normal skin fibroblasts. The promoter regions are highlighted with red boxes. n = 2. ( B ) ATAC-Seq analysis for the immune checkpoints CD47 and PD-L1 and the interleukin IL-6 in scleroderma fibroblasts, scleroderma fibroblasts after JUN knockout or under vismodegib, and normal skin fibroblasts. The promoter regions are highlighted with red boxes. n = 2. ( C ) Heatmap of differential open chromatin regulatory elements characterized from ATAC-Seq. The color bar shows the relative ATAC-Seq signal ( Z score of normalized read counts) as indicated. Samples 1 and 2 in both groups are individual samples. n = 2. ( D ) Fibrosis-linked genes with a 5-fold decline in promoter accessibility after JUN knockout. n = 2. ( E ) p-JUN expression in pulmonary fibroblasts and scleroderma fibroblasts on a 70 kPa hydrogel or a regular polystyrene plastic dish. Two-sided t test. ** P < 0.01; *** P < 0.001. n = 4. Tukey’s multiple comparison test. Bars represent means with standard deviations.

Article Snippet: For immunohistochemistry/immunofluorescence we used adiponectin (Abcam, ab22554), CD3 (Abcam, ab5690), CD11b (Novus, NB110-89474), CD26 (Abcam, ab28340), CD26 (R&D Systems, Bio-Techne, AF954), CD31 (Dako, m0823), CD47 (Thermo Fisher Scientific, 14-0479-82, clone B6H12), CD47 (R&D Systems, Bio-Techne, AF1866), CD68 (Agilent, GA60691-2, clone KP1), cleaved caspase-3 (CST, 96645, clone 5A1E), FSP1 (MilliporeSigma, 07-2274), FSP1 (Abcam, ab58597), collagen 1 (Abcam, ab34710), FSP1 (MilliporeSigma, 07-2274), Ki67 (Abcam, ab15580), PD-1 (Cell Marque, 315M-96, clone NAT105), PD-1 (R&D Systems, Bio-Techne, AF1021), PD-L1 (R&D Systems, Bio-Techne, AF1019), and phospho–c-Jun (Ser73) (CST, 32705, clone D47G9).

Techniques: Knock-Out, Expressing, Comparison

Journal: JCI Insight

Article Title: CD47 prevents the elimination of diseased fibroblasts in scleroderma

doi: 10.1172/jci.insight.140458

Figure Lengend Snippet: ( A ) Immunofluorescence stains against CD47 and FSP1 with and without JUN induction. Scale bar: 25 μm. n = 5. ( B ) Histogram of CD47 expression in fibroblasts with and without JUN induction. n = 5. ( C ) Percentage of CD47 positivity in different fibroblast populations with and without JUN induction. Fisher’s multiple comparison test. ** P < 0.01; *** P < 0.01. n = 5. Bars represent means with standard deviations. ( D ) Representative optical images of ectopically transplanted JUN-inducible mouse dermal fibroblasts ± CD47 inhibition. n = 4. ( E ) Corresponding quantification of photon emissions. Values are normalized to day 0. Fisher’s multiple comparison test. ** P < 0.01. n = 4. Bars represent means with standard deviations. ( F ) Fluorescent graft visualization under the dissection microscope after 7 days of CD47 inhibition. Scale bar: 5 mm. n = 2. ( G ) FACS plot for PE/RFP + CD11b + macrophages ± JUN induction ± CD47 inhibition in an in vitro phagocytosis assay. n = 3. ( H ) Corresponding quantification of RFP + macrophages. Tukey’s multiple comparison test. * P < 0.05; *** P < 0.01. n = 3. Bars represent means with standard deviations. ( I ) Schema of a macrophage depletion trial with subsequent skin fibrosis induction. n = 5. ( J ) Corresponding trichrome stains. Scale bar: 500 μm. n = 5. ( K ) Corresponding hydroxyproline assay. Two-sided t test. *** P < 0.001. n = 5. Bars represent means with standard deviations.

Article Snippet: For immunohistochemistry/immunofluorescence we used adiponectin (Abcam, ab22554), CD3 (Abcam, ab5690), CD11b (Novus, NB110-89474), CD26 (Abcam, ab28340), CD26 (R&D Systems, Bio-Techne, AF954), CD31 (Dako, m0823), CD47 (Thermo Fisher Scientific, 14-0479-82, clone B6H12), CD47 (R&D Systems, Bio-Techne, AF1866), CD68 (Agilent, GA60691-2, clone KP1), cleaved caspase-3 (CST, 96645, clone 5A1E), FSP1 (MilliporeSigma, 07-2274), FSP1 (Abcam, ab58597), collagen 1 (Abcam, ab34710), FSP1 (MilliporeSigma, 07-2274), Ki67 (Abcam, ab15580), PD-1 (Cell Marque, 315M-96, clone NAT105), PD-1 (R&D Systems, Bio-Techne, AF1021), PD-L1 (R&D Systems, Bio-Techne, AF1019), and phospho–c-Jun (Ser73) (CST, 32705, clone D47G9).

Techniques: Immunofluorescence, Expressing, Comparison, Inhibition, Dissection, Microscopy, In Vitro, Phagocytosis Assay, Hydroxyproline Assay

Journal: JCI Insight

Article Title: CD47 prevents the elimination of diseased fibroblasts in scleroderma

doi: 10.1172/jci.insight.140458

Figure Lengend Snippet: ( A ) Schematic outline of the therapeutic trial. ( B ) Representative H&E and trichrome stains of the different groups. Scale bar: 500 μm. n = 4. ( C ) Hydroxyproline content of the skin. Tukey’s multiple comparison test. * P < 0.05; ** P < 0.01. n = 6. Graph bars represent means with standard deviations. ( D ) Amount of fat tissue. Values indicate μm 2 /μm skin width. Tukey’s multiple comparison test. * P < 0.05. n = 8. Graph bars represent means with standard deviations. ( E ) Representative optical images of ectopically transplanted GFP/luciferase-labeled human scleroderma fibroblasts ± CD47/IL-6 inhibition. ( F ) Optical imaging of explanted kidneys on day 5. ( G ) Quantification of photon emissions of explanted kidney grafts normalized to the values of the untreated mice. Two-sided t test. * P < 0.05. n = 3–4. Bars represent means with standard deviations. ( H ) Corresponding caspase-3 staining of kidney grafts. Scale bar: 25 μm. ( I ) Corresponding percentage of caspase-3 + GFP + fibroblasts. Two-sided t test. ** P < 0.01. n = 4–5. Bars represent means with standard deviations.

Article Snippet: For immunohistochemistry/immunofluorescence we used adiponectin (Abcam, ab22554), CD3 (Abcam, ab5690), CD11b (Novus, NB110-89474), CD26 (Abcam, ab28340), CD26 (R&D Systems, Bio-Techne, AF954), CD31 (Dako, m0823), CD47 (Thermo Fisher Scientific, 14-0479-82, clone B6H12), CD47 (R&D Systems, Bio-Techne, AF1866), CD68 (Agilent, GA60691-2, clone KP1), cleaved caspase-3 (CST, 96645, clone 5A1E), FSP1 (MilliporeSigma, 07-2274), FSP1 (Abcam, ab58597), collagen 1 (Abcam, ab34710), FSP1 (MilliporeSigma, 07-2274), Ki67 (Abcam, ab15580), PD-1 (Cell Marque, 315M-96, clone NAT105), PD-1 (R&D Systems, Bio-Techne, AF1021), PD-L1 (R&D Systems, Bio-Techne, AF1019), and phospho–c-Jun (Ser73) (CST, 32705, clone D47G9).

Techniques: Comparison, Luciferase, Labeling, Inhibition, Optical Imaging, Staining

Journal: Nature

Article Title: Exosomes Facilitate Therapeutic Targeting of Oncogenic Kras in Pancreatic Cancer

doi: 10.1038/nature22341

Figure Lengend Snippet: ( A ) Exosomes and liposomes numbers and size distribution-using NanoSight ™ . ( B ) Transmission electron micrograph of exosomes and stained for CD9 by immunogold (left panel: 2ary antibody only), scale bar: 100nm. ( C ) FC analyses for CD63 and CD47 on exosomes (n=3 distinct exosomes isolations). ( D ) FC analyses and quantification of exosomal proteins CD63 and CD47 in liposomes. ( E ) Schematic representation of electroporation of RNAi into exosomes. ( F ) Schematic and fluorescence intensity plot of sucrose gradient layers (from the “Bottom-Up” method, UC: ultracentrifuge). Results from three independent experiments are shown. ( G ) Schematic and fluorescence intensity plot of sucrose gradient layers (from the “Top-Down” method, UC: ultracentrifuge). Results from three independent experiments are shown. The data is presented as the mean ± SEM. FC: Flow cytometry. See accompanying source data.

Article Snippet: For overexpression of CD47 on BJ fibroblasts, transfections were performed using Lipofectamine 2000 reagent (Invitrogen) with pCMV6-AC-GFP CD47 plasmid (Origene, MG204706), after which exosomes were isolated using the standard protocol described below.

Techniques: Transmission Assay, Staining, Electroporation, Fluorescence, Flow Cytometry

Journal: Nature

Article Title: Exosomes Facilitate Therapeutic Targeting of Oncogenic Kras in Pancreatic Cancer

doi: 10.1038/nature22341

Figure Lengend Snippet: ( A ) Fluorescence intensity in sucrose gradient layers from “Bottom-Up” method (see ), ( B ) FC analysis of exosomes with AF647 tagged siRNA in the circulation (n=6 mice per group). ( C ) Quantification of PKH67 labeled exosomes in the indicated organs (n=3 mice) ( D ) FC analyses of pancreas cells 6 hours following injection of siKras G12D Exos (n=5 mice), siKras G12D Lipos (n=5 mice), and PBS (untreated, n=3 mice). ( E ) Quantification of Alexa 647 + /CD11b + monocytes in the blood 3 hours post i.p. injection. One way ANOVA: (Non treated, n=12 mice) vs (Liposomes, n=9 mice), IgG+siKras G12D iExo (n=9 mice), anti-CD47 (B6H12)+siKras G12D iExo (n=8 mice) and anti-CD47 (2D3)+siKras G12D iExo (n=6 mice). Unpaired two-tailed t test: siKras G12D iExo (n=13 mice) and (CD47 high iExo, n=7 mice). Unpaired two-tailed t test: (mouse WT Exosomes, n=9 mice) and (mouse CD47 k/o exosomes, n=9 mice). The mean +/− SEM is depicted. Unless otherwise stated, one-way ANOVA was used. * p<0.05, ** p< 0.01, *** p<0.001, **** p<0.0001. FC: Flow cytometry; mo: mouse. See accompanying source data.

Article Snippet: For overexpression of CD47 on BJ fibroblasts, transfections were performed using Lipofectamine 2000 reagent (Invitrogen) with pCMV6-AC-GFP CD47 plasmid (Origene, MG204706), after which exosomes were isolated using the standard protocol described below.

Techniques: Fluorescence, Labeling, Injection, Two Tailed Test, Flow Cytometry

Journal: Nature

Article Title: Exosomes Facilitate Therapeutic Targeting of Oncogenic Kras in Pancreatic Cancer

doi: 10.1038/nature22341

Figure Lengend Snippet: ( A ) Schematic representation of gating strategy for data shown in . ( B ) FC analysis of CD11b + cells in the circulation, liposomes (n=7 mice), exosomes (n=7 mice), Untreated mice (n=4). ( C ) Representative dot plots from . ( D ) FC analyses of SIRP-α (CD172a) expression from Alexa 647 + /CD11b + monocytes. ( E ) FC analyses of the binding efficiency of CD47 neutralizing antibodies to exosomes (n=3 distinct batches of exosomes). ( F ) Quantification of the number of exosomes/mL in the plasma of WT C57BL/6 mice (n=5) vs. CD47 knockout mice (n=7), unpaired two-tailed t test. The data is presented as the mean ± SEM. Unless otherwise stated, one-way ANOVA was used to determine statistical significance. * p<0.05, *** p<0.001. FC: Flow cytometry. See accompanying source data.

Article Snippet: For overexpression of CD47 on BJ fibroblasts, transfections were performed using Lipofectamine 2000 reagent (Invitrogen) with pCMV6-AC-GFP CD47 plasmid (Origene, MG204706), after which exosomes were isolated using the standard protocol described below.

Techniques: Expressing, Binding Assay, Knock-Out, Two Tailed Test, Flow Cytometry

Journal: Nature

Article Title: Exosomes Facilitate Therapeutic Targeting of Oncogenic Kras in Pancreatic Cancer

doi: 10.1038/nature22341

Figure Lengend Snippet: ( A ) Quantification and representative pictures (scale bar: 100μm) of pancreas structure in KTC mice injected with exosomes with AF647 tagged siRNA, n=3 mice. ( B ) Quantification and representative images (scale bar: 100μm) of pancreas of mice injected with the indicated conditions, n=3 mice, unpaired two-tailed t test. ( C ) Representative images (scale bar: 50μm) for data presented in Fig. 3E–H . ( D ) Quantification of macropinocytic and exosomes uptake (independent experiment, identical statistical analyses). ( E ) AF647 RNAi-tagged exosomes/liposomes uptake in Panc-1 cells (scale bar: 100μm). n=3 independent experiments. ( F ) CM-DiI tagged CD47 k/o vs . WT exosomes uptake in Panc-1 cells (scale bar: 100μm). n=3 independent experiments. ( G ) CM-DiI tagged CD47 k/o vs . WT exosomes uptake in BxPC-3 cells (scale bar: 100μm). n=3 independent experiments. The data is presented as the mean ± SEM. Unless otherwise stated, one-way ANOVA was used to determine statistical significance. ns: not significant. * p<0.05, ** p< 0.01, *** p<0.001, **** p<0.0001. See accompanying source data.

Article Snippet: For overexpression of CD47 on BJ fibroblasts, transfections were performed using Lipofectamine 2000 reagent (Invitrogen) with pCMV6-AC-GFP CD47 plasmid (Origene, MG204706), after which exosomes were isolated using the standard protocol described below.

Techniques: Injection, Two Tailed Test