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: Journal of nanobiotechnology

Article Title: Thrombolytic therapy based on lyophilized platelet-derived nanocarriers for ischemic stroke.

doi: 10.1186/s12951-023-02206-5

Figure Lengend Snippet: Fig. 1 A Schematic representation of the preparation of rtPA-loaded CSM derived from platelets. B Representative mean hydrodynamic diameter of CSM and CSM@rtPA before and after lyophilization (CSM@rtPA/L) process measured by DLS. Data represent mean ± SEM (n = 3, independent samples). C Representative mean diameter of CSM and CSM@rtPA before and after lyophilization process (CSM@rtPA/L) measured by NTA. Data represent mean ± SEM (n = 3, independent samples). D Representative STEM-in SEM images of CSM@rtPA negatively stained with uranyl acetate. Scale bars: 200 nm. E Loading capacity of rtPA encapsulated in CSM samples. Data represent mean ± SEM (n = 3, independent samples). F Representative density plots and quantitative analysis of CSM and CSM@rtPA measured by FC. Scatter density plots of Green fluorescence signal (Green-B channel, rtPA channel) versus Red fluorescence signal (Red-R channel, CellMask DeepRed channel) for CSM, CSM@rtPA, and CSM@rtPA sample after labeling with CellMask Deep Red for lipid staining. Mean fluorescence intensities (MFI) for Green-B channel (D) and Red-R channel. Data represent mean ± SEM (n = 3, independent samples). G. Schematic representation of platelets and platelet-derived CSM surface proteins studied by APC-fluorescently labeled antibodies: anti-hCD47 Ab and anti-hCD42b/GPlba Ab. In vitro Ab binding to platelets and CSM@rtPA before and after lyophilization process. Representative MFI histogram of Red-R channel (APC signal) for platelets, CSM@rtPA and CSM@rtPA/L samples after incubation with APC-anti-CD47 and APC-anti-CD42b/GPIbα antibodies

Article Snippet: Then, fluorescently antibodies, human anti-CD47 APC conjugated Antibody (FAB4670A, R&D Systems) and human antiCD42b/GPlbα APC conjugated Antibody (FAB4067A, R&D systems) were incubated with platelets and with CSMs (10 μL of antibody stock solution /106 cells) and the samples were analyzed by flow cytometer.

Techniques: Derivative Assay, Lyophilization, Staining, Fluorescence, Labeling, In Vitro, Binding Assay, Incubation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Multiparametric characterization of red blood cell physiology after hypotonic dialysis based drug encapsulation process

doi: 10.1016/j.apsb.2021.10.018

Figure Lengend Snippet: Comparison of the amount of each protein detected in RBCs, before and after encapsulation process, and with or without asparaginase (ASNase). The number of copies per cell of the proteins identified in pRBCs, proRBCs, eryaspase ( n = 4 per group) was compared between each pair of samples using a scatter plot. The Pearson correlation coefficient ( R ) was calculated for comparisons between all samples. Data for total and ghosts RBCs were separated. Some key RBCs proteins were displayed: hemoglobin proteins (HBB, HBA1), peroxiredoxin 2 (PRDX2), carbonic anhydrase (CA1, CA2), catalase (CAT), band 3 anion exchanger (SLC4A1), alpha and beta spectrin (SPTA1 and SPTB), ankyrin (ANK1), tropomyosin (TPM3), alpha and beta adducin (ADD1 and ADD2), calpain 1 catalytic subunit (CAPN1), glutathione- S -synthetase (GSS), actin (ACTB), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), Glycophorin A (GYPA), CD47.

Article Snippet: Phosphatidylserine (PS) exposure at the outer membrane leaflet of RBCs and CD47 were assessed using Annexin V-PE (Miltenyi, 130-118-363) and anti-CD47-PE antibody (Miltenyi 130-101-348), respectively.

Techniques: Comparison, Encapsulation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Multiparametric characterization of red blood cell physiology after hypotonic dialysis based drug encapsulation process

doi: 10.1016/j.apsb.2021.10.018

Figure Lengend Snippet: PS exposure, CD47 expression and RBCs-EVs release before and after encapsulation process.

Article Snippet: Phosphatidylserine (PS) exposure at the outer membrane leaflet of RBCs and CD47 were assessed using Annexin V-PE (Miltenyi, 130-118-363) and anti-CD47-PE antibody (Miltenyi 130-101-348), respectively.

Techniques: Expressing, Encapsulation

Journal: Cancers

Article Title: Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stromal Cells as an Efficient Nanocarrier to Deliver siRNA or Drug to Pancreatic Cancer Cells

doi: 10.3390/cancers15112901

Figure Lengend Snippet: Phenotypical characterization of UC-MSCs by flow cytometry (unfilled histogram = CTRL/filled histogram = labeled cells). Histograms show positivity for the tetraspanins CD9, CD63 and CD81, as well as the MSC markers CD73, CD166, CD146, CD105, HLA-ABC, CD47 and CD200, and negativity for the hematopoietic markers HLA-DR and CD45.

Article Snippet: Cells were harvested after detachment with TrypLE Select, washed in PBS (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated for 30 min with the following monoclonal antibodies: CD105-FITC (Ancell corporation, Bayport, NY, USA), CD73-PE (Miltenyi Biotec, Bergisch Gladbach, Germany), CD146-PC5 (Beckman Coulter, Analis, Suarlée, Belgium), CD166-PE (BD Biosciences, Erembodegem, Belgium), CD45-PC7 (BD Biosciences, Erembodegem, Belgium), HLA-ABC- PC5 (BioLegend, San Diego, CA, USA), HLA-DR-PC5 (Beckman Coulter, Analis, Suarlée, Belgium), CD47-APC (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD200-PC7 (BD).

Techniques: Flow Cytometry, Labeling

Journal: Cancers

Article Title: Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stromal Cells as an Efficient Nanocarrier to Deliver siRNA or Drug to Pancreatic Cancer Cells

doi: 10.3390/cancers15112901

Figure Lengend Snippet: Phenotypical characterization of EVs derived from hUC-MSCs by flow cytometry (black line = CTRL, red line = labeled cells). Histograms show positivity for the tetraspanins CD9, CD63 and CD81, as well as for the MSC markers CD73, CD166, CD146, CD105, HLA-ABC, CD47 and CD200, and negativity for the hematopoietic markers HLA-DR and CD45.

Article Snippet: Cells were harvested after detachment with TrypLE Select, washed in PBS (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated for 30 min with the following monoclonal antibodies: CD105-FITC (Ancell corporation, Bayport, NY, USA), CD73-PE (Miltenyi Biotec, Bergisch Gladbach, Germany), CD146-PC5 (Beckman Coulter, Analis, Suarlée, Belgium), CD166-PE (BD Biosciences, Erembodegem, Belgium), CD45-PC7 (BD Biosciences, Erembodegem, Belgium), HLA-ABC- PC5 (BioLegend, San Diego, CA, USA), HLA-DR-PC5 (Beckman Coulter, Analis, Suarlée, Belgium), CD47-APC (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD200-PC7 (BD).

Techniques: Derivative Assay, Flow Cytometry, Labeling

Journal: Molecular Therapy Oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: CD47 levels in human lung adenocarcinoma cells were increased following tumor metastasis (A and B) Comparison of CD47 expression between primary lung adenocarcinoma and lung adenocarcinoma metastases in the lymph nodes. (C and D) Comparison of CD47 expression between primary lung adenocarcinoma and lung adenocarcinoma metastases in the liver. (A) and (C) showed representative IHC images for CD47 staining from 14 to 5 lung adenocarcinoma tissue samples, respectively. Data were presented as means ± SDs. ∗∗p < 0.01. Scale bar, 50 μm.

Article Snippet: Primary antibodies against CD47 (AF4670, R&D Systems, Minneapolis, MN, USA), PD-L1 (#13684, Cell Signaling Technology) and IRF1 (11335-1-AP, Proteintech) were used.

Techniques: Comparison, Expressing, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Induction of CD47 by IFN-γ in human lung cancer cells (A) Flow cytometry analysis of CD47 surface expression in human lung cancer cell lines upon incubation with recombinant IFN-γ (100 ng/mL, 24 h). Right: Representative image; left: quantitative analysis. (B) Immunofluorescence analysis of CD47 expression in human lung cancer cell lines upon incubation with recombinant IFN-γ (100 ng/mL, 24 h). Scale bar, 20 μm. (C) Western blot analysis of CD47 expression in human lung cancer cell lines after IFN-γ treatment. (D) qRT-PCR analysis of CD47 mRNA in human lung cancer cell lines upon incubation with recombinant IFN-γ (100 ng/mL, 24 h). Left: Representative image; right: quantitative analysis. Data from more than 3 independent experiments were presented as means ± SDs. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Primary antibodies against CD47 (AF4670, R&D Systems, Minneapolis, MN, USA), PD-L1 (#13684, Cell Signaling Technology) and IRF1 (11335-1-AP, Proteintech) were used.

Techniques: Flow Cytometry, Expressing, Incubation, Recombinant, Immunofluorescence, Western Blot, Quantitative RT-PCR

Journal: Molecular Therapy Oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Identification of genes involved in the IFN signaling pathway that upregulates CD47 expression (A) qRT-PCR analysis of various genes involved in the IFN signaling pathway in A549 cells upon incubation with recombinant IFN-γ (100 ng/mL, 24 h). (B) Normalized luciferase reporter expression of A549 cells transduced with different sets of lentiviral shRNA constructs. Upper: Schematic representation of the CD47-Prom-Firefly Luciferase-EF1AProm-Renilla luciferase constructs used to generate the reporter cell lines. Lower: Quantitative analysis. (C) IRF1 protein levels in human lung cancer cells upon incubation with recombinant IFN-γ (100 ng/mL, 24 h). Upper: Representative image; lower: quantitative analysis. Data from more than 3 independent experiments were presented as means ± SDs. ∗∗∗p < 0.001.

Article Snippet: Primary antibodies against CD47 (AF4670, R&D Systems, Minneapolis, MN, USA), PD-L1 (#13684, Cell Signaling Technology) and IRF1 (11335-1-AP, Proteintech) were used.

Techniques: Expressing, Quantitative RT-PCR, Incubation, Recombinant, Luciferase, Transduction, shRNA, Construct

Journal: Molecular Therapy Oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: IFN-γ induces CD47 expression in lung cancer cells through IRF-1 (A) Sequence of the CD47 promoter showing the position of the putative IRF-1-binding site. (B) Reporter assay for putative IRF-1 binding. The results were normalized to relative luciferase units (RLUs). (C) ChIP assay analyzing the CD47 promoter in A549 cells. The results were normalized to the input. (D and E) IRF-1 mRNA (D) and protein (E) levels in A549 cells transfected with IRF-1-specific shRNA or scramble shRNA after IFN-γ treatment. (F and G) CD47 level in A549 cells transfected with IRF-1-specific shRNA or scramble shRNA after IFN-γ treatment detected by flow cytometry (F) and immunofluorescence (G). Scale bar, 10 μm. (H) Western blot analysis of CD47 levels in A549 cells transfected with IRF-1-specific or scramble shRNA. Left: Representative image; right: quantitative analysis. Data from 3 independent experiments were presented as means ± SDs. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Primary antibodies against CD47 (AF4670, R&D Systems, Minneapolis, MN, USA), PD-L1 (#13684, Cell Signaling Technology) and IRF1 (11335-1-AP, Proteintech) were used.

Techniques: Expressing, Sequencing, Binding Assay, Reporter Assay, Luciferase, Transfection, shRNA, Flow Cytometry, Immunofluorescence, Western Blot

Journal: Molecular Therapy Oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: IFN-γ promotes lung cancer cell metastasis by upregulating CD47 expression in vitro (A) Scratch-wound healing assay in A549 cells (A549-WT) and CD47-knockout A549 cells (A549-CD47-KO) were treated with/without IFN-γ. (B) Transwell assay in A549 cells (A549-WT) and A549-CD47-KO cells were treated with/without IFN-γ. Data from 3 independent experiments were presented as means ± SDs. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Scale bar, 200 μm.

Article Snippet: Primary antibodies against CD47 (AF4670, R&D Systems, Minneapolis, MN, USA), PD-L1 (#13684, Cell Signaling Technology) and IRF1 (11335-1-AP, Proteintech) were used.

Techniques: Expressing, In Vitro, Wound Healing Assay, Knock-Out, Transwell Assay

Journal: Molecular Therapy Oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: IFN-γ promotes human lung cancer cell metastasis in immunodeficient mice by upregulating CD47 (A) A549 cells (A549-WT) and A549-CD47-KO were engrafted in the lungs of the BALB/c-nude mice. Three weeks post-engraftment, the mice were randomly divided into 2 groups. One group was administered with IFN-γ (10 ng/mouse, injected once every 2 days) (A549-WT + IFN-γ; A549-CD47-KO + IFN-γ), and the other group without IFN-γ injection was served as control (A549-WT; A549-CD47-KO). After 5 weeks, the mice were sacrificed to analyze tumor growth and metastasis. (B and C) H&E staining and human CD47 immune staining in mouse lungs (B) and livers (C). In both (B) and (C), left: representative images; right: quantification of tumor nodules. Data were presented as means ± SDs. ∗p < 0.05, ∗∗∗p < 0.001. NS, no significance.

Article Snippet: Primary antibodies against CD47 (AF4670, R&D Systems, Minneapolis, MN, USA), PD-L1 (#13684, Cell Signaling Technology) and IRF1 (11335-1-AP, Proteintech) were used.

Techniques: Injection, Control, Staining

Journal: Oncology Letters

Article Title: CD47 as a potential prognostic marker for oral leukoplakia and oral squamous cell carcinoma

doi: 10.3892/ol.2018.8520

Figure Lengend Snippet: Expression of CD47 in oral squamous cell carcinoma cell lines and normal oral keratinocytes, as assessed by immunofluorescence. CD47 was expressed in (A1) Tca8113, (B1) Cal-27 and (C1) SCC-9 cells. (D1) Weak positive staining was observed in normal oral keratinocytes. No expression of CD47 was observed in the control cells (A2-D2; magnification, ×400). CD47, cluster of differentiation 47.

Article Snippet: The sections were then incubated at 4°C overnight in a diluted solution (dilution, 1:200) of monoclonal mouse anti-human CD47 (cat. no. MAB4670; R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Expressing, Immunofluorescence, Staining, Control

Journal: Oncology Letters

Article Title: CD47 as a potential prognostic marker for oral leukoplakia and oral squamous cell carcinoma

doi: 10.3892/ol.2018.8520

Figure Lengend Snippet: Effect of CD47 antibody on the proliferation of Cal-27 cells. The proliferation of Cal-27 cells was inhibited by 5 and 10 µg/ml CD47 antibody. *P<0.05, **P<0.01 and ***P<0.001 vs. control group. CD47, cluster of differentiation 47; OD, absorbance; CCK-8, Cell Counting kit-8.

Article Snippet: The sections were then incubated at 4°C overnight in a diluted solution (dilution, 1:200) of monoclonal mouse anti-human CD47 (cat. no. MAB4670; R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Control, CCK-8 Assay, Cell Counting

Journal: Nature Communications

Article Title: Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing

doi: 10.1038/s41467-022-31386-1

Figure Lengend Snippet: Schematic presentation of different parallel or anti-parallel coiled coil pairing. N5N6 (strong parallel CC pair), P3AP4 (anti-parallel CC pair) and P3SP4S (weak parallel CC pair) ( a ). Diagram showing gRNA positions within exon 2 of the human CD47 gene ( b ). Indel mutation quantification by the T7E1 assay, determined 48 h post transfection (HEK293 cells (2*10 5 cells/ml) were transfected with plasmids encoding gRNA and CCExo) for several tested gRNAs ( c – h ), a combination of all gRNAs ( i ), and gRNA1 in combination with CCExo containing coiled-coils with increasing affinity (P3P4 < N5N6) ( j ). Data present three individual separate experiments ( n = 3). * P < 0.05, **< 0.01, ***< 0.001 and **** P < 0.0001. All P values are from ordinary one-way ANOVA followed by Tukey’s multiple comparisons test compared to Cas9 values or if stated otherwise by brackets. Data are presented as mean values ± SEM as appropriate ( c – j ). Expression of Cas9 or its variants in HEK293 cells 48 h post transfection determined by western blot and immunodetection using specific anti-Cas9 antibodies. α-tubulin was used as a loading control. A representative blot from two individual separate experiment is shown ( k ).

Article Snippet: For CD47 detection we used human CD47-FITC antibodies from Miltenyi Biotec (130-101-344).

Techniques: Mutagenesis, Transfection, Expressing, Western Blot, Immunodetection

Journal: Nature Communications

Article Title: Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing

doi: 10.1038/s41467-022-31386-1

Figure Lengend Snippet: HEK293 cells (2*10 5 cells/ml) were transfected with plasmids encoding target gRNA and CCExo. Genomic DNA was isolated 48 h post transfection, the targeted region PCR amplified and subjected to NGS analysis. NGS analysis was performed for 200 bp amplicons for gRNA targeting MYD88 gene ( a ) or 400 bp amplicons for gRNA targeting CD47 gene ( b ), VEGFα gene ( c ) or EMX1 gene ( d ). Diagrams are showing percentage (calculated based on number of total mutated reads) of deletions (Δ) of various lengths. Genome wide determinations of potential off-target activities of CCExo for VEGFα sites in HEK293 cells and for TRBC1 site in human K562 cells, predicted by CIRCLE-seq ( e ).

Article Snippet: For CD47 detection we used human CD47-FITC antibodies from Miltenyi Biotec (130-101-344).

Techniques: Transfection, Isolation, Amplification, Genome Wide