Journal: Journal of Neuroinflammation

Article Title: Menopause leads to elevated expression of macrophage-associated genes in the aging frontal cortex: rat and human studies identify strikingly similar changes

doi: 10.1186/1742-2094-9-264

Figure Lengend Snippet: Age- and ovarian hormone-related changes in expression of genes related to microglial reactivity in the frontal cortex of middle-aged female rats

Article Snippet: Cd47 , Rn00569914_m1 , 0.884 , 0.084 , 1.189 a , 0.041 , 1.051 , .

Techniques: Expressing, TaqMan Assay

Journal: Journal of Neuroinflammation

Article Title: Menopause leads to elevated expression of macrophage-associated genes in the aging frontal cortex: rat and human studies identify strikingly similar changes

doi: 10.1186/1742-2094-9-264

Figure Lengend Snippet: Age- and ovarian hormone-dependent regulation of genes encoding microglial receptors and their inhibitory neuronal ligands in the rat frontal cortex. TaqMan-based quantitative real-time PCR revealed no change in Sirpa ( A ), increase in the expression Cd47 ( B ), decrease in mRNA expression of Cd200r ( C ), Cd200 ( D ) and Cx3cl1 ( F ). Cx3cr1 ( E ) increased during aging. Error bars show SD of six independent measurements. Statistical significance of the alterations in different groups compared to middle-aged female rats (M) was determined by analysis of variance (ANOVA) with Newman-Keuls post hoc test, and considered at P <0.05. Asterisks indicate changes with statistical significance: *corresponds to 0.01< P < 0.05, ** to 0.001< P < 0.01 and *** to P < 0.001. Y, young rat; M/OVX, middle-aged OVX rat; M/OVX+E2, middle-aged ovariectomized (OVX) rat treated with 17β-estradiol (E2).

Article Snippet: Cd47 , Rn00569914_m1 , 0.884 , 0.084 , 1.189 a , 0.041 , 1.051 , .

Techniques: Real-time Polymerase Chain Reaction, Expressing

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: Nature Communications

Article Title: Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection

doi: 10.1038/s41467-024-47963-5

Figure Lengend Snippet: HBECs were infected with ( + IAV ) or without ( Mock ) influenza A virus. a Immunoblot analysis of junction protein ZO-1 and surface protein CD47 at 1 day post-infection (dpi). Normalized CD47 and ZO-1 protein levels are presented as bar graphs (Mock, n = 4; + IAV, n = 4). b Representative whole-mount images of ZO-1 (white) and CD47 (red) at 1 dpi. The area where ZO-1 disconnection occurred are presented as bar graphs (Mock, n = 6; + IAV, n = 6). c Quantitative PCR (qPCR) analysis of ZO-1 and CD47 mRNAs at 1 and 3 dpi [Mock, n = 3; + IAV (1 dpi), n = 3; + IAV (3 dpi), n = 3]. d HBECs were treated with or without 10 μM NF-κB inhibitor caffeic acid phenethyl ester (CAPE) for 1 h before influenza virus infection. Immunoblot analysis of ICAM-1 and CD47 at 1 dpi. Normalized ICAM-1 and CD47 protein levels are presented as bar graphs (DMSO + Mock, n = 4; DMSO + IAV, n = 4; CAPE + Mock, n = 4; CAPE + IAV, n = 4). e Whole-mount images of influenza virus-infected HBECs. Co-staining of CD47 (red) and ciliated cell-specific marker protein Ac-α-tubulin (green, n = 4) or goblet cell-specific marker protein MUC5AC (cyan, n = 3). Percentages of CD47-positive cells are presented as bar graphs. Data are presented as mean values ± SEM. Significance was determined by unpaired two-tailed Student’s t test or one-way ANOVA with Tukey’s multiple comparisons test. n.s. not significant. Source data are provided as a Source Data file.

Article Snippet: On days 5 and 7 after viral infection, mice were injected intranasally with either IgG2a control antibodies (clone 2A3; Cat.BE0089; BioXCell) or anti-mouse CD47 antibodies (clone MIAP301; Cat.BE0270; BioXCell).

Techniques: Infection, Virus, Western Blot, Real-time Polymerase Chain Reaction, Staining, Marker, Two Tailed Test

Journal: Nature Communications

Article Title: Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection

doi: 10.1038/s41467-024-47963-5

Figure Lengend Snippet: For CD47 knock-down, HBECs were transfected with scrambled shRNA ( sc-shRNA ) or shRNA targeting CD47 ( CD47 shRNA ) using a lentiviral delivery system. For CD47 neutralization, HBECs were treated with either IgG1 (MOPC-21) or α-hCD47 (B6H12.2) antibodies. a Gene expression of CD47 was analyzed using qRT-PCR (normalized by GAPDH mRNA) (sc-shRNA, n = 4; CD47 shRNA, n = 4). b Protein expression of CD47 was analyzed using immunoblotting (sc-shRNA, n = 5; CD47 shRNA, n = 5). c Paracellular FITC-dextran permeability was measured in Mock ( n = 16), Virus only (IAV, n = 16), Bacteria only ( S. aureus , n = 16), sc-shRNA + Super-infection ( n = 12), CD47 shRNA + Super-infection ( n = 6), IgG1 + Super-infection ( n = 6), and α-hCD47 antibodies (Ab) + Super-infection ( n = 8). d Trans-epithelial resistance was measured in Mock ( n = 15), IAV ( n = 14), S. aureus ( n = 13), sc-shRNA + Super-infection ( n = 13), CD47 shRNA + Super-infection ( n = 6), IgG1 + Super-infection ( n = 6), and α-hCD47 Ab + Super-infection ( n = 8). e Microscopic images of HBECs at 5 dpi. The percentage of the damaged area is presented as bar graphs (Mock, n = 3; Virus only, n = 3; Bacteria only, n = 3; Super-infection, n = 4; IgG1 + Super-infection, n = 4; α-hCD47 Ab + Super-infection, n = 4). Data are presented as mean values ± SEM. Significance was determined by unpaired two-tailed Student’s t test or one-way ANOVA with Tukey’s multiple comparisons test. N.D. not determined. Source data are provided as a Source Data file.

Article Snippet: On days 5 and 7 after viral infection, mice were injected intranasally with either IgG2a control antibodies (clone 2A3; Cat.BE0089; BioXCell) or anti-mouse CD47 antibodies (clone MIAP301; Cat.BE0270; BioXCell).

Techniques: Transfection, shRNA, Neutralization, Expressing, Quantitative RT-PCR, Western Blot, Permeability, Virus, Bacteria, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection

doi: 10.1038/s41467-024-47963-5

Figure Lengend Snippet: a , b Influenza virus-infected HBECs ( IAV ) infected with live S. aureus or S. aureus -derived samples ( S , supernatant of S. aureus -cultured media; U , UV-killed S. aureus ; H , heat-killed S. aureus ). Paracellular FITC-dextran permeability ( a ) and trans-epithelial electrical resistance ( b ) of 7 groups: i ) Mock ( n = 3), ii ) Virus only ( n = 3), iii ) Bacteria only ( n = 3), iv ) Super-infection ( n = 3), v ) Virus with S ( n = 3), vi ) Virus with U ( n = 3), and vii ) Virus with H ( n = 3). c Whole mount image of CD47 (red) and S. aureus (green) in the influenza virus-infected HBECs at 1 dpi. An open arrow head indicates CD47 + cell without S. aureus and closed arrow heads indicate CD47 + cells with S. aureus . Co-localization of CD47 + cells and S. aureus are presented as violin plots (Mock, n = 6; + IAV, n = 6). d – g Bacterial adhesion assay. Colonization of S. aureus was assessed in HBECs inoculated with the virus (MOI 1) for 2 h before treatment with IgG1 control antibodies or α-hCD47 neutralizing antibodies (2 h), followed by S. aureus (MOI 3) infection for 3 h (IgG1 + S. aureus , n = 4; α-hCD47 Ab + S. aureus , n = 4; IgG1 + Super-infection, n = 4; α-hCD47 Ab+ Super-infection, n = 4) ( d ). Adherence of S. aureus WT (FnBP A+/B+, n = 4) and three mutant strains (FnBP A+/B–, n = 4; FnBP A–/B+, n = 4; FnBP A–/B–, n = 4) was assessed in HBECs inoculated with the virus (MOI 1) for 2 h, followed by S. aureus (MOI 3) infection for 3 h ( e ). Colonization of S. aureus WT (FnBP A+/B+) ( f ) and double deletion mutant (FnBP A–/B–) ( g ) was assessed in HBECs inoculated with the virus (MOI 1) for 2 h before treatment with IgG1 control antibodies ( n = 4) or α-hCD47 neutralizing antibodies (1, 5, and 10 μg/mL for FnBP A+/B+, and 10 μg/mL for FnBP A–/B–, n = 4 each) for 2 h, followed by S. aureus (MOI 3) infection for 3 h. h Pull-down assay using His-tagged hCD47 recombinant protein. Bacterial plating [FnBP A+/B+ and FnBP A–/B– ( n = 3 each in the absence or in the presence of His-tagged hCD47)] and immunoblot analysis were performed using supernatants and pellets after separation with α-His-Dynabeads™/DynaMag™−2 system. The graphs present the percentage of colony numbers grown in the culture of the supernatants or the pellets. Data are presented as mean values ± SEM. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test or unpaired two-tailed Student’s t test. n.s. not significant. Source data are provided as a Source Data file.

Article Snippet: On days 5 and 7 after viral infection, mice were injected intranasally with either IgG2a control antibodies (clone 2A3; Cat.BE0089; BioXCell) or anti-mouse CD47 antibodies (clone MIAP301; Cat.BE0270; BioXCell).

Techniques: Virus, Infection, Derivative Assay, Cell Culture, Permeability, Bacteria, Cell Adhesion Assay, Mutagenesis, Pull Down Assay, Recombinant, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection

doi: 10.1038/s41467-024-47963-5

Figure Lengend Snippet: 6–8-weeks-old (18–21 g of body weight) FoxJ1 -Cre;floxed ( CD47 Foxj1 ), LysM -Cre;floxed ( CD47 LysM ), and control floxed ( CD47 f/f ) mice were infected with 100 PFU of influenza virus on day 0, and 1 ×10 8 CFU of S. aureus on day 7. a , b , k , l Body weight loss ( a , k ) and survival rates ( b , l ) were monitored for 29 days. The dotted line indicates the body weight exclusion cut-off. A mantel cox survival analysis was used to compare the survival rates between groups. The numbers in parenthesis represent the count of surviving mice. c , m Representative hematoxylin and eosin (H&E) staining of lung sections. The dotted lines indicate lymphocytic infiltration and arrows indicate alveolar hemorrhage. Lung injury scores are presented as violin plots in CD47 Foxj1 mice ( CD47 f/f , n = 8; CD47 Foxj1 n = 5) ( c ) and CD47 LysM mice ( CD47 f/f , n = 8; CD47 LysM , n = 8) ( m ). d – j , n – t Tissue injury parameters were measured at 24 h after bacterial infection; total cell number in BAL fluids (BALF) of CD47 Foxj1 mice ( CD47 f/f , n = 5; CD47 Foxj1 , n = 5) ( d ) and CD47 LysM mice ( CD47 f/f , n = 8; CD47 LysM , n = 10) ( n ); bacterial adherence in the lung of CD47 Foxj1 mice ( CD47 f/f , n = 10; CD47 Foxj1 , n = 11) ( e ) and CD47 LysM mice ( CD47 f/f , n = 13; CD47 LysM , n = 15) ( o ); bacterial invasion in the lung of CD47 Foxj1 mice ( CD47 f/f , n = 7; CD47 Foxj1 , n = 7) ( f ) and CD47 LysM mice ( CD47 f/f , n = 5; CD47 LysM , n = 5) ( p ); and bacterial burden in the spleen of CD47 Foxj1 mice ( CD47 f/f , n = 5; CD47 Foxj1 , n = 6) ( g ) and CD47 LysM mice ( CD47 f/f , n = 5; CD47 LysM , n = 5) ( q ); total protein concentrations in BALF of CD47 Foxj1 mice ( CD47 f/f , n = 5; CD47 Foxj1 , n = 5) ( h ) and CD47 LysM mice ( CD47 f/f , n = 8; CD47 LysM , n = 10) ( r ); and inflammatory cytokines TNF-α in BALF of CD47 Foxj1 mice ( CD47 f/f , n = 5; CD47 Foxj1 , n = 5) ( i ) and CD47 LysM mice ( CD47 f/f , n = 8; CD47 LysM , n = 10) ( s ), and IL-6 in BALF of CD47 Foxj1 mice ( CD47 f/f , n = 5; CD47 Foxj1 , n = 5) ( j ) and CD47 LysM mice ( CD47 f/f , n = 8; CD47 LysM , n = 10) ( t ). Data are presented as mean values ± SEM. Significance was determined by unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.

Article Snippet: On days 5 and 7 after viral infection, mice were injected intranasally with either IgG2a control antibodies (clone 2A3; Cat.BE0089; BioXCell) or anti-mouse CD47 antibodies (clone MIAP301; Cat.BE0270; BioXCell).

Techniques: Infection, Virus, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection

doi: 10.1038/s41467-024-47963-5

Figure Lengend Snippet: For neutralization test, 6–8-weeks-old (18–21 g of body weight) C57BL/6 WT mice were infected with 10 PFU of influenza virus on day 0 and 5 × 10 5 CFU of S. aureus on day 7. Before bacterial infection, mice were intranasally treated twice at day 5 and 7 with IgG2a control antibodies (2A3, n = 17) or α-mCD47 neutralizing antibodies (MIAP301, n = 16). a , b Body weight loss ( a ) and survival rates ( b ) were monitored for 29 days. The dotted line indicates the body weight exclusion cut-off. A mantel cox survival analysis was used to compare the survival rates between groups. The numbers in parenthesis are numbers of survived mice. c Representative hematoxylin and eosin (H&E) staining of lung sections (IgG2a + Mock, n = 4; α-mCD47 Ab + Mock, n = 4; IgG2a + Virus only, n = 7; α-mCD47 Ab + Virus only, n = 7; IgG2a + Bacteria only, n = 4; α-mCD47 Ab + Bacteria only, n = 4; IgG2a + Super-infection, n = 9; α-mCD47 Ab + Super-infection, n = 11). The dotted lines indicate lymphocytic infiltration and arrows indicate alveolar hemorrhage. Lung injury scores are presented as violin plots. d – j Tissue injury parameters were measured at 24 h after bacterial infection (IgG2a + Mock, n = 4; α-mCD47 Ab + Mock, n = 4; IgG2a + Virus only, n = 7; α-mCD47 Ab + Virus only, n = 7; IgG2a + Bacteria only, n = 4; α-mCD47 Ab + Bacteria only, n = 4; IgG2a + Super-infection, n = 7-9; α-mCD47 Ab + Super-infection, n = 7-11); total cell number in BAL fluids (BALF) ( d ), bacterial adherence ( e ) and invasion ( f ) in the lung, and the bacterial burden in the spleen ( g ), total protein concentrations ( h ) in BALF, and inflammatory cytokines TNF-α ( i ) and IL-6 ( j ) in BALF. Data are presented as mean values ± SEM. Significance was determined by unpaired two-tailed t test. n.s. not significant. N.D. not determined. Source data are provided as a Source Data file.

Article Snippet: On days 5 and 7 after viral infection, mice were injected intranasally with either IgG2a control antibodies (clone 2A3; Cat.BE0089; BioXCell) or anti-mouse CD47 antibodies (clone MIAP301; Cat.BE0270; BioXCell).

Techniques: Neutralization, Infection, Virus, Staining, Bacteria, Two Tailed Test

Journal: Nature Communications

Article Title: Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection

doi: 10.1038/s41467-024-47963-5

Figure Lengend Snippet: a – j 6–8-weeks-old (18–21 g of body weight) FoxJ1 -Cre; floxed ( Cd47 Foxj1 ) and control floxed ( Cd47 f/f ) mice were infected with 100 PFU of influenza virus on day 0, and 1 ×10 8 CFU of S. aureus WT (FnBP A+/B+) and double deletion mutant (FnBP A–/B–) on day 7 ( Cd47 f/f + FnBP A+/B+, n = 9; Cd47 f/f + FnBP A–/B–, n = 11; Cd47 Foxj1 + FnBP A+/B+, n = 12; Cd47 Foxj1 +FnBP A–/B–, n = 10). Body weight loss ( a ) and survival rates ( b ) were monitored for 29 days. The dotted line indicates the body weight exclusion cut-off. A mantel cox survival analysis was used to compare the survival rates between groups. The numbers within circles or squares represent the count of surviving mice. Representative hematoxylin and eosin (H&E) staining of lung sections ( c ). Lung injury scores are presented as violin plots ( Cd47 f/f + FnBP A+/B+, n = 4; Cd47 f/f + FnBP A–/B–, n = 5; Cd47 Foxj1 + FnBP A+/B+, n = 5; Cd47 Foxj1 +FnBP A–/B–, n = 5). Tissue injury parameters were measured at 24 h after bacterial infection ( Cd47 f/f + FnBP A+/B+, n = 4; Cd47 f/f + FnBP A–/B–, n = 5; Cd47 Foxj1 + FnBP A+/B+, n = 5; Cd47 Foxj1 +FnBP A–/B–, n = 5); total cell number in BAL fluids (BALF) ( d ), bacterial adherence ( e ) and invasion ( f ) in the lung, and bacterial burden in the spleen ( g ), total protein concentrations in BALF ( h ), and inflammatory cytokines TNF-α ( i ) and IL-6 ( j ) in BALF. k Proposed model of viral infection-induced CD47-mediated secondary bacterial infection. Data are presented as mean values ± SEM. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

Article Snippet: On days 5 and 7 after viral infection, mice were injected intranasally with either IgG2a control antibodies (clone 2A3; Cat.BE0089; BioXCell) or anti-mouse CD47 antibodies (clone MIAP301; Cat.BE0270; BioXCell).

Techniques: Infection, Virus, Mutagenesis, Staining

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: SARS-CoV-2 infection is associated with increased CD47 levels. A) TF protein abundance in uninfected (control) and SARS-CoV-2-infected (virus) Caco-2 cells (data derived from . P-values were determined by two-sided Student’s t-test. B) CD47 and SARS-CoV-2 N protein levels and virus titres (genomic RNA determined by PCR) in SARS-CoV-2 strain FFM7 (MOI 1)-infected air-liquid interface cultures of primary human bronchial epithelial (HBE) cells and SARS-CoV-2 strain FFM7 (MOI 0.1)-infected Calu-3 cells. Uncropped blots are provided in Suppl. Figure 1. C) CD47 mRNA levels in post mortem samples from COVID-19 patients (data derived from ). P-values were determined by two-sided Student’s t-test.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Infection, Quantitative Proteomics, Control, Virus, Derivative Assay

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 aging” (A) and “CD47 hypertension” (B). C) Overview figure of the data derived from the literature searches. Age-related increased CD47 levels may contribute to pathogenic conditions associated with severe COVID-19.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 diabetes” (A). B) Overview figure of the data derived from the literature search. Hyperglycaemia- and diabetes-induced increased CD47 levels may contribute to immune escape of SARS-CoV-2-infected cells.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Derivative Assay, Infection

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: Translational Oncology

Article Title: Regulation of CD47 expression by interferon-gamma in cancer cells

doi: 10.1016/j.tranon.2021.101162

Figure Lengend Snippet: Primers and siRNA sequences.

Article Snippet: The primary antibodies used to detect the targeting proteins expression were as follows: CD47 (Cell Signaling Technology (CST), Beverly, USA, #63000), JAK1 (CST, #3332S), JAK2 (CST, #3230S), STAT1 (CST, #14994), p-STAT1 (Tyr701) (CST, #9167), IRF1 (CST, #8478S) and GAPDH (CST.

Techniques: Negative Control

Journal: Translational Oncology

Article Title: Regulation of CD47 expression by interferon-gamma in cancer cells

doi: 10.1016/j.tranon.2021.101162

Figure Lengend Snippet: IFN-γ up-regulated CD47 expression. In A549 and NCI-H1975 cells, RT-PCR was to detect mRNA expression of CD47 (a) , western blot was applied to detect the CD47 protein expression (b) , flow cytometry was used to detect cell surface expression of CD47 (c) . ( d) After treatment with IFN-γ (10 ng/ml) for 3, 6, 12, 24 h in two cancer cell lines, the surface protein expression of CD47 was detected by flow cytometry, respectively. *, P < 0.05; **, P < 0.01; ns, nonsignificant.

Article Snippet: The primary antibodies used to detect the targeting proteins expression were as follows: CD47 (Cell Signaling Technology (CST), Beverly, USA, #63000), JAK1 (CST, #3332S), JAK2 (CST, #3230S), STAT1 (CST, #14994), p-STAT1 (Tyr701) (CST, #9167), IRF1 (CST, #8478S) and GAPDH (CST.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry

Journal: Translational Oncology

Article Title: Regulation of CD47 expression by interferon-gamma in cancer cells

doi: 10.1016/j.tranon.2021.101162

Figure Lengend Snippet: Ruxolitinib inhibited CD47 up-regulation induced by IFN-γ. A549 and NCI-H1975 cells were pretreated with ruxolitinib (1 μM) for 1 hours followed by treatment with IFN-γ (10 ng/ml) for 24 h. The mRNA level of CD47 was detected by RT-PCR (a) , Immunofluorescence was used to detected CD47 expression in different treatment groups. The Hoechst 33342 was to stain the cell nuclei and anti-CD47 antibody was to stain CD47 protein. Scale bar: 250 μm ( b), Western blot analysis showed p-STAT1 expression and CD47 expression ( c) , The surface protein expression of CD47 was determined by flow cytometry (d). ( e) After A549 cells were treated with or without ruxolitinib, co-culture experiment was conducted by flow cytometry and then the relatively phagocytosis index was calculated. *, P < 0.05; **, P < 0.01.

Article Snippet: The primary antibodies used to detect the targeting proteins expression were as follows: CD47 (Cell Signaling Technology (CST), Beverly, USA, #63000), JAK1 (CST, #3332S), JAK2 (CST, #3230S), STAT1 (CST, #14994), p-STAT1 (Tyr701) (CST, #9167), IRF1 (CST, #8478S) and GAPDH (CST.

Techniques: Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Expressing, Staining, Western Blot, Flow Cytometry, Co-Culture Assay

Journal: Translational Oncology

Article Title: Regulation of CD47 expression by interferon-gamma in cancer cells

doi: 10.1016/j.tranon.2021.101162

Figure Lengend Snippet: Knockdown of JAK1 significantly inhibits CD47 up-regulation induced by IFN-γ. (a-b) After successfully knockdown of JAK1 or JAK2, the cell surface CD47 expression with or without IFN-γ exposure was detected by flow cytometry in A549 and NCI-H1975 cells. ( c) The JAK1/2 and CD47 expression was showed by western blot after knockdown of JAK1/2 with or without IFN-γ treatment. ( d) SIRPα binding assay was conducted to exhibit the binding ability of CD47 to SIRPα in A549 cells after silenced JAK1. ( e) In A549 cells and BMDMs co-culture system, the phagocytosis index changes after knockdown JAK1 combined with or without IFN-γ were analyzed by flow cytometry. *, P < 0.05; **, P < 0.01.

Article Snippet: The primary antibodies used to detect the targeting proteins expression were as follows: CD47 (Cell Signaling Technology (CST), Beverly, USA, #63000), JAK1 (CST, #3332S), JAK2 (CST, #3230S), STAT1 (CST, #14994), p-STAT1 (Tyr701) (CST, #9167), IRF1 (CST, #8478S) and GAPDH (CST.

Techniques: Knockdown, Expressing, Flow Cytometry, Western Blot, Binding Assay, Co-Culture Assay

Journal: Translational Oncology

Article Title: Regulation of CD47 expression by interferon-gamma in cancer cells

doi: 10.1016/j.tranon.2021.101162

Figure Lengend Snippet: Knockdown of STAT1 inhibited CD47 up-regulation induced by IFN-γ. (a) The protein expression of STAT1 and CD47 was analyzed by western blot after knockdown of STAT1 by siRNA in A549 and NCI-H1975 cells. ( b) The cell surface CD47 expression was revealed by flow cytometry after knockdown of STAT1 with or without IFN-γ treatment. (c) Flow cytometry was used to detect the surface expression of CD47 after the STAT1 inhibitor fludarabine treatment. (d) After knockdown of STAT1, the binding of CD47 to SIRPα was determined by flow cytometry in A549 cells. (e) The relative phagocytosis index of BMDMs to A549 cells was calculated based on flow cytometry result. *, P < 0.05; **, P < 0.01.

Article Snippet: The primary antibodies used to detect the targeting proteins expression were as follows: CD47 (Cell Signaling Technology (CST), Beverly, USA, #63000), JAK1 (CST, #3332S), JAK2 (CST, #3230S), STAT1 (CST, #14994), p-STAT1 (Tyr701) (CST, #9167), IRF1 (CST, #8478S) and GAPDH (CST.

Techniques: Knockdown, Expressing, Western Blot, Flow Cytometry, Binding Assay

Journal: Translational Oncology

Article Title: Regulation of CD47 expression by interferon-gamma in cancer cells

doi: 10.1016/j.tranon.2021.101162

Figure Lengend Snippet: Knockdown of IRF1 inhibited CD47 up-regulation induced by IFN-γ. (a) The protein expression of IRF1 and CD47 was analyzed by western blot analysis after knockdown of IRF1 by siRNA in A549 and NCI-H1975 cells. ( b) Surface expression of CD47 was detected by flow cytometry after silenced IRF1 in two lung cancer cell lines. ( c) SIRPα binding assay was presented to showed CD47 binding ability to SIRPα after knockdown of IRF1 in A549 cells. ( d) Co-culture A549 cells with BMDMs, the phagocytosis index change was measured based on flow cytometry result. *, P < 0.05; **, P < 0.01.

Article Snippet: The primary antibodies used to detect the targeting proteins expression were as follows: CD47 (Cell Signaling Technology (CST), Beverly, USA, #63000), JAK1 (CST, #3332S), JAK2 (CST, #3230S), STAT1 (CST, #14994), p-STAT1 (Tyr701) (CST, #9167), IRF1 (CST, #8478S) and GAPDH (CST.

Techniques: Knockdown, Expressing, Western Blot, Flow Cytometry, Binding Assay, Co-Culture Assay

Journal: Translational Oncology

Article Title: Regulation of CD47 expression by interferon-gamma in cancer cells

doi: 10.1016/j.tranon.2021.101162

Figure Lengend Snippet: IFN-γ induced CD47 up-regulation was a widespread phenomenon in cancer cells. (a) The correlations between CD47 and IFNG in different cancer cell types were analyzed by calculating the Pearson correlation coefficients. ( b) In different cancer cell lines, the up-regulation of CD47 induced by IFN-γ was detected by flow cytometry. *, P < 0.05; **, P < 0.01.

Article Snippet: The primary antibodies used to detect the targeting proteins expression were as follows: CD47 (Cell Signaling Technology (CST), Beverly, USA, #63000), JAK1 (CST, #3332S), JAK2 (CST, #3230S), STAT1 (CST, #14994), p-STAT1 (Tyr701) (CST, #9167), IRF1 (CST, #8478S) and GAPDH (CST.

Techniques: Flow Cytometry

Journal: Molecular and Clinical Oncology

Article Title: Systemic immune responses are associated with molecular characteristics of circulating tumor cells in head and neck squamous cell carcinoma

doi: 10.3892/mco.2021.2309

Figure Lengend Snippet: Association between systemic immune-related markers and molecular characteristics of CTCs in patients (n=28).

Article Snippet: Primers for the 14 target genes epithelial cell adhesion molecule [ EPCAM : Hs00158980_m1], MET : Hs01565576_m1, keratin 19 [ KRT19 : Hs00761767_s1], epidermal growth factor receptor [ EGFR : Hs01076090_m1], phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha [ PIK3CA : Hs00907957_m1], cyclin D1 [ CCND1 : Hs00765553_m1], snail family transcriptional repressor 1 [ SNAI1 : Hs00195591_m1], vimentin [ VIM : Hs00958111_m1], CD44 : Hs01075861_m1, nanog homeobox [ NANOG : Hs04399610_g1], aldehyde dehydrogenase 1 family member A1 [ ALDH1A1 : Hs00946916_m1], CD47 : Hs00179953_m1, CD274 : Hs01125301_m1, and programmed cell death 1 ligand 2 [ PDCD1LG2 : Hs01057777_m1] and ACTB (Hs01060665_g1, normalization control) were purchased from Applied Biosystems (TaqManTM Gene Expression Assays).

Techniques: