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: 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: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Histograms depicting cell surface expression of CD24 and CD47 by flow cytometry on mouse cancer cell lines. B, Correlation of CD24 and CD47 surface expression of cell lines shown in A by geometric MFI. Data shown as mean ± SD of 3 technical replicates. Simple linear regression was performed to assess correlation. C, Representative plots showing quantification of CD45+ phagocytic primary mouse macrophages co-cultured with CFSE+ KPCA.C. Co-cultures were exposed to vehicle control (PBS) or 10 ug/ml of monoclonal antibodies against mouse CD47, CD24, or the combination for 2 hours. Phagocytosis is represented as CD45+ macrophages that had engulfed CFSE+ KPCA.C cells as a percentage of the total macrophage population. D, Quantification of phagocytosis for cell lines in A . Cell lines are organized based on expression levels of each surface marker. Data represent mean ± SD of 3 technical replicates. E, Correlation of cell surface expression levels of CD47 and CD24 compared to phagocytosis upon treatment with the corresponding antibodies for each cell line. Data points depict mean ± SD from 3 replicates for each experiment. Correlation was assessed by simple linear regression. F, Representative microscopy images of GFP+ KPCA.C cells when co-cultured with primary mouse macrophages upon treatment with vehicle control (PBS), 10 ug/mL anti-CD47, 10 ug/mL anti-CD24, or the combination for 6.5 days. Top row depicts raw images of GFP+ fluorescence. Bottom row depicts purple GFP+ mask for above images used for quantification of cancer cell growth. Scale bar, 800 µm. G, Quantification of fluorescent well area from co-culture experiments for multiple cell lines after 6.5 days, organized by surface expression of CD24. Cancer cells were quantified by either green (KPCA.C, 3LL ΔNRAS, MC38) or red (238N1) fluorescent area based on their fluorophore expression. Data and means shown from one (3LL ΔNRAS, MC38) or two (238N1, KPCA.C) independent experiments with 3 technical replicates per experiment. D,G, statistical significance ns, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 determined by two-way ANOVA with Holm-Sidak multiple comparison test.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Expressing, Flow Cytometry, Cell Culture, Control, Bioprocessing, Marker, Microscopy, Fluorescence, Co-Culture Assay, Comparison

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Representative histograms demonstrating cell surface expression of CD47 and CD24 on knockouts of KPCA.C and knockdowns of 238N1 by flow cytometry. B, Representative gating of phagocytic APC CD45+ mouse macrophages when co-cultured with the indicated CFSE+ KPCA.C knockouts treated with vehicle control (PBS) for 2 hours. Phagocytic macrophages are calculated as CD45+ cells that have engulfed CFSE+ cancer cells after 2 hours as a percent of all macrophages. C,D, Quantification of phagocytosis as a percentage of the maximum phagocytic response of macrophages using KPCA.C knockout cells ( C ) or 238N1 knockdown cells ( D ) treated with vehicle control (PBS), anti-mouse CD47 antibody, anti-mouse CD24 antibody, or the combination. Data represents mean ± SD of 3 technical replicates. E,F, Quantification of fluorescent well area as a measure of GFP+ KPCA.C knockout cells ( E ) or mCherry+ 238N1 knockdown cells ( F ) growth after co-culture with primary mouse macrophages and the indicated antibodies on day 6.5. Data represents mean ± SD from two independent experiments of 3 technical replicates each. G,H, Quantification of phagocytosis using CFSE+ MC38 ( G ) or 3LL ΔNRAS ( H ) cancer cells that overexpress CD24 after co-culture with primary mouse macrophages and the indicated antibodies. Data represent mean ± SD from 3 individual experiments each containing 3 technical replicates. I, Quantification of phagocytosis using StayGold+ KPCA.C cancer cells treated with vehicle control (PBS), or anti-mouse CD24 antibody, in the absence or presence of FcR blocking reagents (Fc1, anti-mouse Truestain clone 93; Fc2, anti-mouse CD16/CD32 clone 2.4G2). Data represents mean ± SD of 3 technical replicates. ( C-H ) ns, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA with Holm-Sidak multiple comparison test.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Expressing, Flow Cytometry, Cell Culture, Control, Knock-Out, Knockdown, Co-Culture Assay, Blocking Assay, Comparison

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: Results of scRNA-seq of sorted CD45+ immune cells from experiments using CD24 or CD47 knockout tumors. ( A,C,E ) Comparison of CD47- tumors (KPCA.C CD47 knockout, 238N1 CD47 knockout) to wild-type tumors (KPCA.C control, 238N1 control). A, Relative frequencies of immune cells from CD47- versus wild-type tumors. C, UMAP showing identified cell clusters. E, Gene set enrichment analysis showing Normalized Enrichment Scores of top Hallmark pathways. ( B,D,F ) Comparison of CD24- tumors (KPCA.C CD24 knockout, 238N1 CD24 knockout) to wild-type tumors (KPCA.C control, 238N1 control). B, Relative frequencies of immune cells from CD24- versus wild-type tumors. D, UMAP showing identified cell clusters. F, Gene set enrichment analysis showing Normalized Enrichment Scores of top Hallmark pathways.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Knock-Out, Comparison, Control

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Diagram showing process for high-throughput development and functional evaluation of bispecific antibodies targeting macrophage immune checkpoints. Antibody sequences were transformed into scFvs and cloned into a knob-into-hole format using a human IgG1 Fc. Constructs targeting macrophage immune checkpoints (CD47, CD24, SIRPa, PD-1) were cloned into knob formats and crossed with tumor-binding constructs in a hole format. Bispecific antibodies (n = 77) were expressed in Expi293F cells and used for downstream biochemical and functional analysis. B, Growth of StayGold+ DLD-1 cells in co-culture with human macrophages and each bispecific antibody. Each curve represents the mean for an individual bispecific antibody from 4 replicates. Black curve with hashed lines represents mean and 95% CI of control wells . C, Anti-tumor efficacy of bispecific antibodies at approximately t = 6.5 days as evaluated by macrophage checkpoint category. *p<0.05, ****p<0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. D-F, Growth curves for each of the WTa2d1 constructs ( D ), CD24-3 constructs ( E ), or CV1 constructs ( F ). G, Representative whole-well imaging of co-cultures treated with different bispecific antibodies at approximately t = 6.5 day. Green signal depicts growth of StayGold+ DLD-1 cells. Rows contain different macrophage checkpoint arms, while columns contain different tumor-binding arms. H, Scatter plot showing binding of each bispecific antibody to human neutrophils versus red blood cells. I, Representative histograms showing binding of the indicated bispecific antibodies to human neutrophils and red blood cells.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: High Throughput Screening Assay, Functional Assay, Transformation Assay, Clone Assay, Construct, Binding Assay, Co-Culture Assay, Control, Imaging

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Surface CD47 and CRT expression in EGFR wild-type and mutant NSCLC cells. Surface CD47 (A) and ecto-CRT protein expression (B) shown as geometric MFI in a panel of six different NSCLC cell lines. Each histogram represents the mean (± SD) of three to five independent experiments. Comparisons made by ANOVA with Fisher's post hoc multiple comparison analysis. ### p < 0.03, ## p < 0.01, # p < 0.0005. Below each histogram, a matrix table where all p values resulting from post hoc analysis are reported. Expression levels of CD47 (C) and CRT mRNA (D) in 226 untreated primary NSCL adenocarcinomas (GEO accession number GSE31210 ). Middle lines in box plots represent the medians and whiskers represent 5–95% CI ( ### p < 0.03, Kruskal-Wallis test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Mutagenesis, Comparison

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Modulation by gefitinib of surface CD47 and CRT expression in EGFR wild-type and mutant NSCLC cells. Flow cytometric profiles of surface CD47 (A) and ecto-CRT expression (B) on DMSO-treated (CTRL, gray lines) and gefitinib-treated (GEF, red lines) NSCLC cells. Histograms show the mean (± SD) of fold changes of CD47 (C) and ecto-CRT (D) geometric MFI, relative to DMSO-treated controls ( N = 3–5, * p < 0.05, ** p < 0.01 paired two-tailed Student's t -test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Mutagenesis, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Gefitinib-induced CD47 down-regulation promotes tumor cell phagocytosis by dendritic cells. Representative flow cytometric analyses and mean ± SD ( N = 4 independent healthy donors) of phagocytic activity of monocyte-derived dendritic cells (see Methods) against PC9 (A,B) , HCC827 (C,D) , and H1975 cells (E,F) treated with DMSO (CTRL) or gefitinib (GEF) as indicated. Cancer cells exposed to the drug for 48 h were labeled with DiO tracer and then co-cultured with dendritic cells for 2 h at a 1:1 ratio. Phagocytosis assays were also run at 4°C as controls. Histograms represent the percentages of positive cells for both CD11c and DiO tracer relative to total dendritic cells (* p < 0.05, n.s., not significant, paired two-tailed Student's t -test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Activity Assay, Derivative Assay, Labeling, Cell Culture, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Blocking of CD47 on tumor cells induces phagocytosis by dendritic cells. Dendritic cells were co-cultured with DiO tracer-labeled HCC827 (A) and H1975 (B) cancer cells in the presence of IgG isotype control or anti-CD47 mAb as indicated. Shown is the mean (± SD, N = 3 independent healthy donors) percentage increase of CD11c/DiO tracer double positive cells, relative to dendritic cells co-cultured with DMSO-treated tumor cells ( # p < 0.05, ## p < 0.01, ANOVA with Fisher's post hoc analysis).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Blocking Assay, Cell Culture, Labeling, Control

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Expression levels of surface CD47 increase in cancer cells acquiring resistance to gefitinib and inhibit tumor cell phagocytosis by dendritic cells. Surface CD47 (A) and ecto-CRT expression (B) in gefitinib-sensitive PC9 and HCC827 (gray lines) and resistant PC9GR and HCC827GR (green lines) cell lines. Representative flow cytometric histograms (left) and mean (± SD, N = 3–5) fold changes of treatment-resistant over sensitive cells (right). (C) Representative flow cytometric histogram plots (left) and mean (± SD) fold changes (right) of surface CD47 levels in resistant cell lines treated with DMSO (CTRL) or gefitinib (GEF) as indicated. Acquisition of resistance to gefitinib abolished drug-induced CD47 down-regulation in PC9GR (* p < 0.05, ** p < 0.01, n.s., not significant, paired two-tailed Student's t -test). (D) Mean ± SD ( N = 3 independent healthy donors) of phagocytic activity of monocyte-derived dendritic cells against PC9GR cells in the absence or presence of gefitinib treatment, performed at 4°C as control and at 37°C. Histograms represent the percentages of positive cells for both CD11c and DiO tracer relative to total dendritic cells (paired two-tailed Student's t -test. n.s., not significant). (E) Dendritic cells were co-cultured with gefitinib-treated, DiO tracer-labeled PC9GR cells in the presence of IgG isotype control or anti-CD47 mAb. Shown is the mean ± SD ( N = 3 independent healthy donors) percent change of CD11c + /DiO + tracer double positive dendritic cells, relative to dendritic cells co-cultured with DMSO-treated tumor cells ( ## p < 0.01, ANOVA with Fisher's post hoc analysis).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Two Tailed Test, Activity Assay, Derivative Assay, Control, Cell Culture, Labeling

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: CD47 is overexpressed on malignant lymphocytes in mycosis fungoides (MF) tumors. a A representative image of a skin involved by MF demonstrates intense CD47 staining on atypical TOX + malignant cells. HE, hematoxylin and eosin (20x). b Targeted single-cell RNA transcriptomics as tSNE plots of concatenated tumors from three patients with MF tumors (n = 287 cells total). Clusters called by recursive dendrogram split and annotated from preferentially expressed genes. Tcm, T cell central memory; Tem, T cell effecor memory; DC, dendritic cells. c The intensity of CD47 expression (anti-CD47 antibody-oligo conjugate; AbSeq) over the various cell population defined in Fig. 1b. d Statistical analysis of expression of CD47 (molecules per cells) in different cell populations. *, p < 0.05; ***, p < 0.001 e CD47 expression on CD3 + TOX + MBL2 cells. Flow cytometry of a cell suspension from a primary cell culture. Grey tinted area, an isotype control. Red tinted area, anti-CD47 antibody. f Tumor growth curves of CD47hi WT MBL2 (WT) and CD47 KO MBL2 (CD47 KO) after implantation in B6.SJL mice. n = 5 mice in each group. g Representative imaging of mice 10 days after implantation of CD47hi WT MBL2 (WT) or CD47 KO MBL2 (KO) cells demonstrating large ulcerated tumor in WT, while KO mouse exhibited medium-size tumor without ulceration

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Staining, Expressing, Flow Cytometry, Suspension, Cell Culture, Control, Imaging

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: Anti-CD47 therapy is efficient in controlling the malignant lymphoma growth in a murine model of mycosis fungoides (MF). a Design of anti-CD47 experiments (I.P., intraperitoneal). b Tumor thickness after MBL2 implantation during treatment with anti-CD47 antibody or irrelevant IgG control (n = 5 mice per group). c Percentage of TOX + tumor cells in the inflammatory infiltrate of auricular skin at day 24 after implantation. The percentage of caspase-3 + cells is indicated in black (n = 5 mice per group) (Tx, treatment). d Percentage of F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). e Percentage of MHC class II + cells among F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). f Percentage of TNF-α + cells among F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). g Percentage of NK1.1 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). h Percentage of IFN-γ + cells among NK1.1 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). i Percentage of CD62L + NKG2A-cells among NK1.1. + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). j Schematic of experimental design. k Representative images of mice with tumors after treatment with IFN-α, anti-CD47, or combination anti-CD47 + IFN-α. l The percentage of malignant cells (TOX +) in the TME on day 24 after implantation of MBL2 (n = 5 mice per group). m Representative flow cytometry of CD107a and IFN-γ NK cells (gated on NK1.1 + cells) during therapy with anti-CD47 antibody. n Quantification of CD107a + IFN-γ + NK cells and CD107a + IFN-γ-NK cells (n = 5 mice per group). o The cytotoxic assay of NK cells derived from splenocytes of treated mice co-cultured with MBL2 cells at the indicated effector to target cell ratios

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Control, Flow Cytometry, Derivative Assay, Cell Culture

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: The effect of anti-CD47 therapy is mediated by cytotoxic NK cells and does not depend on IFN-γ. a Schematic of NK1.1 depletion experiment. b A representative flow showing the percentage of NK cells in non-depleted and NK-depleted mice prior to therapy. c Representative images of mice with tumors 14 days after MBL2 implantation with and without NK1.1 depletion prior to therapy initiation. D The percentage of malignant cells (CD3 + TOX + cells) per ear in non-depleted and NK-depleted mice 24 days after MBL2 implantation. N = 5 mice per group. ***, p < 0.001 e Tumor thickness 24 days after MBL2 implantation in mice treated with irrelevant IgG or anti-CD47 antibody (n = 5 mice per group). f Schematic of experimental design for IFN-γ KO mice. g The volume of lymph nodes 24 days after MBL2 implantation in mice treated with anti-CD47 antibody (n = 3 mice per group). *,p < 0.05; **,p < 0.01; ****,p < 0.0001; ns, non-significant

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques:

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: The anti-CD47 therapy is accompanied by an influx of NK cells in the TME in patients with relapsed/refractory mycosis fungoides (MF). a Representative images of high and low CD56 number in the dermal infiltrate of tumor MF. b Inverse correlation of the number of CD56 cells per 100 dermal lymphocytes and histoscore of CD47 on the epidermotropic malignant lymphocytes (n = 17, r = – 0.48, p < 0.05). c Representative images of a patient treated with six intra-tumoral injections of 10 mg TTI-621 demonstrate a significant reduction of all tumors and plaques after treatment (I, injected tumor; C, control non-injected tumor). d Multispectral fluorescent immunohistochemistry of CD3, CD56, TOX, and DAPI staining of representative tumors before and after treatment with TTI-621 (60X). e Percentage of malignant cells (CD3 + CD4 + TOX +) and NK cells (CD3-CD56 +) in the TME (n = 11 patients; 4 responders and 7 non-responders). f tSNE analysis of cellular composition in patients with cutaneous T cell lymphoma who had responded to therapy with TTI-621 (n = 4 patients), showing eight distinct clusters of NK cells. g Phenotypic characterization of eight distinct clusters of NK cells depending on the percentage of markers. h Grouping of eight NK cell clusters in two groups based on high vs. dim CD56 expression. i Changes in CD56high NK cells vs. CD56dim NK cells after intra-tumoral injection of TTI-621 (2 weeks of therapy) in patients who responded to TTI-621 (n = 4). j Percentage of NK cells before and after six intra-tumoral injections of TTI-621 (2 weeks of therapy) in patients who responded to TTI-621 (n = 4).. *,p < 0.05; **,p < 0.01

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Injection, Control, Immunohistochemistry, Staining, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Aqueous extract of Taxus chinensis var. mairei targeting CD47 enhanced antitumor effects in non-small cell lung cancer.

doi: 10.1016/j.biopha.2022.113628

Figure Lengend Snippet: Fig. 2. AETC enhanced the inhibitory effects of CD47 on LUAD cells. (A/B) Cell viability was examined by CCK-8 assay. (C/D) CD47 mRNA and protein levels after treatment with various AETC concentrations for 24 h. (D) CD47 expression in LUAD cell membranes was evaluated by flow cytometry. (E) CD47 localization was determined by immunofluorescence after AETC treatment for 24 h. Scale bar: 20 µm/50 µm. Data are expressed as mean ± SD; n = 3 independent experiments.

Article Snippet: The following reagents, kits, and antibodies were used: CD45 (No. 103139, BioLegend, San Diego, CA, USA), CD11b (No. 101205, BioLegend), F4/80 (No. 70-AM048005–100, BioLegend), CD68 (No. 333813, BioLegend), CD8 (No. 100733, BioLegend), CD4 (No. 100407, BioLegend), CD3 (No. 100203, BioLegend), CD47 No. (No. 127507, BioLegend), PD-1 (No. 135217, BioLegend), Zombie NIRTM Fixable Viability Kit (No. 423105, BioLegend), Mouse Regulatory T Cell Staining Kit (No. KTR201–25, MultiSciences [LIANKE] Biotech Co. Ltd., Hangzhou, China), MG-132 (No. S2619, Selleck Chemicals), chloroquine (No. S6999, Selleck Chemicals), bortezomib (No. 5043140001, SigmaAldrich Corp., St. Louis, MO, USA), anti-CD47 antibody (No. NBP2–31106, Novus Biologicals, Centennial, CO, USA), InVivoPlus antimouse PD-1 (CD279) (No. BP0033–2, Bio X Cell, Lebanon, NH, USA), collagenase D (No. 11088858001, Roche Diagnostics, Basel, Switzerland), DNase 1 (No. 10104159001, Sigma-Aldrich Corp.), and carboxyfluorescein diacetate succinimidyl ester (CFDA SE) (No. C0051, Beyotime Biotechnology, Shanghai, China).

Techniques: CCK-8 Assay, Expressing, Flow Cytometry, Immunofluorescence

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Aqueous extract of Taxus chinensis var. mairei targeting CD47 enhanced antitumor effects in non-small cell lung cancer.

doi: 10.1016/j.biopha.2022.113628

Figure Lengend Snippet: Fig. 3. AETC-mediated CD47 potently elicited LUAD cell phagocytosis. (A/C/E) Representative images of macrophages phagocytosing LUAD cells after AETC treatment (0.13 or 1 mg/mL) for 24 h. (B/D/F) Effects of AETC (0.13 or 1 mg/mL) on macrophage-mediated phagocytosis of LLC and HCC827 cells were evaluated by flow cytometry. The part circled by the black box (F4/80+/CD68+ CFDA SE+) indicates LUAD cells phagocytosed by macrophages. Differences in the phagocytic indices are shown in the right histogram. *P < 0.05, * * P < 0.01 vs. control group. Data are expressed as mean ± SD; n = 3 independent experiments.

Article Snippet: The following reagents, kits, and antibodies were used: CD45 (No. 103139, BioLegend, San Diego, CA, USA), CD11b (No. 101205, BioLegend), F4/80 (No. 70-AM048005–100, BioLegend), CD68 (No. 333813, BioLegend), CD8 (No. 100733, BioLegend), CD4 (No. 100407, BioLegend), CD3 (No. 100203, BioLegend), CD47 No. (No. 127507, BioLegend), PD-1 (No. 135217, BioLegend), Zombie NIRTM Fixable Viability Kit (No. 423105, BioLegend), Mouse Regulatory T Cell Staining Kit (No. KTR201–25, MultiSciences [LIANKE] Biotech Co. Ltd., Hangzhou, China), MG-132 (No. S2619, Selleck Chemicals), chloroquine (No. S6999, Selleck Chemicals), bortezomib (No. 5043140001, SigmaAldrich Corp., St. Louis, MO, USA), anti-CD47 antibody (No. NBP2–31106, Novus Biologicals, Centennial, CO, USA), InVivoPlus antimouse PD-1 (CD279) (No. BP0033–2, Bio X Cell, Lebanon, NH, USA), collagenase D (No. 11088858001, Roche Diagnostics, Basel, Switzerland), DNase 1 (No. 10104159001, Sigma-Aldrich Corp.), and carboxyfluorescein diacetate succinimidyl ester (CFDA SE) (No. C0051, Beyotime Biotechnology, Shanghai, China).

Techniques: Flow Cytometry, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Aqueous extract of Taxus chinensis var. mairei targeting CD47 enhanced antitumor effects in non-small cell lung cancer.

doi: 10.1016/j.biopha.2022.113628

Figure Lengend Snippet: Fig. 4. AETC suppresses CD47 protein through ubiquitin degradation. (A) CD47 interaction network analyzed in the BioGrid database (https://thebiog rid.org). (B/C) Volcano plot, cluster heatmaps, and enrichment analysis of DEGs were analyzed on the Dr. Tom BGI network platform. (D) Western blotting was performed to detect the CD47 protein levels in LLC and HCC827 cells subjected either to CHX (25 μg/ mL) or CHX plus AETC (0.13 or 1 mg/ mL) for the indicated amount of time. (E) The CD47 protein levels was measured after pretreatment with 10 μM MG-132, 10 μM CQ, or 10 nM bortezomib for 1 h and co-treated with AETC (0.13 or 1 mg/mL) for 24 h. (F) The LLC and HCC827 cells were pre treated with 10 μM MG-132 for 1 h, treated with or without AETC (0.13 or 1 mg/mL) for 24 h, and lysed with NP40 lysis buffer. Immunoprecipitation of cell lysate using the indicated anti body and analysis of immune complex by western blotting using the indicated antibodies. *P < 0.05, **P < 0.01. Data are expressed as mean ± SD; n = 3 in dependent experiments.

Article Snippet: The following reagents, kits, and antibodies were used: CD45 (No. 103139, BioLegend, San Diego, CA, USA), CD11b (No. 101205, BioLegend), F4/80 (No. 70-AM048005–100, BioLegend), CD68 (No. 333813, BioLegend), CD8 (No. 100733, BioLegend), CD4 (No. 100407, BioLegend), CD3 (No. 100203, BioLegend), CD47 No. (No. 127507, BioLegend), PD-1 (No. 135217, BioLegend), Zombie NIRTM Fixable Viability Kit (No. 423105, BioLegend), Mouse Regulatory T Cell Staining Kit (No. KTR201–25, MultiSciences [LIANKE] Biotech Co. Ltd., Hangzhou, China), MG-132 (No. S2619, Selleck Chemicals), chloroquine (No. S6999, Selleck Chemicals), bortezomib (No. 5043140001, SigmaAldrich Corp., St. Louis, MO, USA), anti-CD47 antibody (No. NBP2–31106, Novus Biologicals, Centennial, CO, USA), InVivoPlus antimouse PD-1 (CD279) (No. BP0033–2, Bio X Cell, Lebanon, NH, USA), collagenase D (No. 11088858001, Roche Diagnostics, Basel, Switzerland), DNase 1 (No. 10104159001, Sigma-Aldrich Corp.), and carboxyfluorescein diacetate succinimidyl ester (CFDA SE) (No. C0051, Beyotime Biotechnology, Shanghai, China).

Techniques: Ubiquitin Proteomics, Western Blot, Lysis, Immunoprecipitation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Aqueous extract of Taxus chinensis var. mairei targeting CD47 enhanced antitumor effects in non-small cell lung cancer.

doi: 10.1016/j.biopha.2022.113628

Figure Lengend Snippet: Fig. 5. Synergistic effects of anti-PD-1 plus AETC on tumor-bearing C57BL/6 mice. (A) Body weights in each group. (B) Tumor volumes were recorded every 2 d during treatment. (C) Images of harvested tumors. (D) Mice were sacrificed after 14 d treatment and the tumor quality inhi bition rates were calculated. (E) Inhibitory effects of anti-PD-1 plus AETC on CD47. (F) Inhibitory effects of anti-PD-1 plus AETC on PD-1. *P < 0.05, **P < 0.01. Data are expressed as mean ± SD; n = 3 independent experiments.

Article Snippet: The following reagents, kits, and antibodies were used: CD45 (No. 103139, BioLegend, San Diego, CA, USA), CD11b (No. 101205, BioLegend), F4/80 (No. 70-AM048005–100, BioLegend), CD68 (No. 333813, BioLegend), CD8 (No. 100733, BioLegend), CD4 (No. 100407, BioLegend), CD3 (No. 100203, BioLegend), CD47 No. (No. 127507, BioLegend), PD-1 (No. 135217, BioLegend), Zombie NIRTM Fixable Viability Kit (No. 423105, BioLegend), Mouse Regulatory T Cell Staining Kit (No. KTR201–25, MultiSciences [LIANKE] Biotech Co. Ltd., Hangzhou, China), MG-132 (No. S2619, Selleck Chemicals), chloroquine (No. S6999, Selleck Chemicals), bortezomib (No. 5043140001, SigmaAldrich Corp., St. Louis, MO, USA), anti-CD47 antibody (No. NBP2–31106, Novus Biologicals, Centennial, CO, USA), InVivoPlus antimouse PD-1 (CD279) (No. BP0033–2, Bio X Cell, Lebanon, NH, USA), collagenase D (No. 11088858001, Roche Diagnostics, Basel, Switzerland), DNase 1 (No. 10104159001, Sigma-Aldrich Corp.), and carboxyfluorescein diacetate succinimidyl ester (CFDA SE) (No. C0051, Beyotime Biotechnology, Shanghai, China).

Techniques:

Journal: Cell Reports Medicine

Article Title: CD47-blocking antibody confers metabolic benefits against obesity

doi: 10.1016/j.xcrm.2025.102089

Figure Lengend Snippet: Administration of CD47-blocking antibody confers metabolic protective effects in mice under HFD condition (A) Correlation of CD47 expression with BMI in individuals with type 2 diabetes (T2D). (B) Correlation between CD47 expression and HOMA-IR in obese individuals. (C and D) CD47 mRNA and protein expression levels in skeletal muscle of control and obese mice ( n = 4). Expression levels quantified, GAPDH as control. (E and F) CD47 mRNA and protein expression in sedentary and exercise-trained mice after 8 weeks ( n = 4). Expression quantified, GAPDH as control. (G) Schematic of antibody injection protocol: CD47-blocking antibody (CD47 Ab) or control IgG antibody (Cont) every two days under HFD. (H) Bodyweight changes in mice injected with CD47 Ab or Cont on HFD ( n = 6 for Cont and n = 7 for CD47 Ab). (I and J) Body composition measured by NMR in mice ( n = 6 for Cont and n = 8 for CD47 Ab). (K) Intraperitoneal glucose tolerance test (IPGTT) and area under the curve (AUC) after 5 weeks HFD administration ( n = 6). (L–N) Oxygen consumption, carbon dioxide emissions, and energy expenditure measured using metabolic cage ( n = 5 for Cont and n = 6 for CD47 Ab). (O and P) Spontaneous food intake and physical activity ( n = 5 for Cont and n = 6 for CD47 Ab). (Q and R) Running distance and duration measured on a treadmill ( n = 6). Data are presented as means ± SEM and analyzed by two-tailed Student’s t test (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant).

Article Snippet: Milk (5%) in TBST was used to block members for 1 h at room temperature, then the members were washed with TBST three times (5 min each time) and incubated with primary antibodies against P-AMPK (Thr172, 2535), AMPK (2532), P-HSP90α (Thr5/7, 3488), P-ACC (3661), ACC (3676), p-DNA-PK (Ser2056, 68716), COXIV (4850), cyto C (4280), Acetylated-Lysine (9814) (Cell Signaling Technology Inc.), SIRT1 (04-1557, Millipore), PGC-1α (ab54481, Abcam), HSP90α (A5006, ABclonal), DNA-PK (ab70250, Abcam), AMPKγ3 (A14132, ABclonal), AMPKγ1 (A22024, ABclonal), CD47 (AF1866-SP, R&D systems), and GAPDH (AB0037, Abways) in 5% BSA at 4°C overnight.

Techniques: Blocking Assay, Expressing, Control, Injection, Activity Assay, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: CD47-blocking antibody confers metabolic benefits against obesity

doi: 10.1016/j.xcrm.2025.102089

Figure Lengend Snippet: CD47-blocking antibody promotes skeletal muscle AMPK activation and improves mitochondrial function (A) AMPK phosphorylation in skeletal muscle, WAT, BAT, liver, hypothalamus (Hypo), and kidney ( n = 6). (B) ACC phosphorylation in skeletal muscle after antibody injection. GAPDH as control. (C) PGC-1α protein levels in skeletal muscle post-injection. GAPDH as control. (D) Western blot analysis of AMPK, ACC phosphorylation, and PGC-1α in skeletal muscle of mice injected with CD47 Ab (20 mg/kg), Cont (20 mg/kg), and AMPK inhibitor (Compound C, 10 mg/kg) ( n = 3). (E and F) AMPK phosphorylation and cytochrome c (cyto c) protein expression in myotubes after antibody treatment. GAPDH as control. (G) Seahorse mitochondrial stress test in myotubes treated with CD47 Ab. (H–K) AMPK, ACC phosphorylation, and PGC-1α expression in myotubes with Cd47 knockdown or overexpression. (L) Seahorse mitochondrial stress test in myotubes with Cd47 knockdown. Data are presented as means ± SEM and analyzed by two-tailed Student’s t test (A and G) and two-way repeated measures (RM) ANOVA (D and L). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant.

Article Snippet: Milk (5%) in TBST was used to block members for 1 h at room temperature, then the members were washed with TBST three times (5 min each time) and incubated with primary antibodies against P-AMPK (Thr172, 2535), AMPK (2532), P-HSP90α (Thr5/7, 3488), P-ACC (3661), ACC (3676), p-DNA-PK (Ser2056, 68716), COXIV (4850), cyto C (4280), Acetylated-Lysine (9814) (Cell Signaling Technology Inc.), SIRT1 (04-1557, Millipore), PGC-1α (ab54481, Abcam), HSP90α (A5006, ABclonal), DNA-PK (ab70250, Abcam), AMPKγ3 (A14132, ABclonal), AMPKγ1 (A22024, ABclonal), CD47 (AF1866-SP, R&D systems), and GAPDH (AB0037, Abways) in 5% BSA at 4°C overnight.

Techniques: Blocking Assay, Activation Assay, Phospho-proteomics, Injection, Control, Western Blot, Expressing, Knockdown, Over Expression, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: CD47-blocking antibody confers metabolic benefits against obesity

doi: 10.1016/j.xcrm.2025.102089

Figure Lengend Snippet: CD47 LOF in skeletal muscle promotes AMPK activation (A) Schematic of CD47-knockout (CD47 −/− ) mouse model. (B) Growth curves of WT and CD47 −/− mice ( n = 6). (C) IPGTT and AUC in WT and CD47 −/− mice ( n = 6). (D and E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes in skeletal muscle of CD47 −/− and exercise mice. (F) AMPK and ACC phosphorylation in skeletal muscle of WT and CD47 −/− mice. (G) PGC-1α, cyto c, and oxidase (COXIV) expression levels in skeletal muscle of WT and CD47 −/− mice. (H and I) ATPase and CS activities in skeletal muscle of WT and CD47 −/− mice ( n = 7). (J) Schematic of MCK-CD47 mouse model. (K) Growth curves of MCK-CD47 mice on HFD ( n = 10 for Con and n = 6 for MCK-CD47). (L) IPGTT and AUC in MCK-CD47 mice after 3 months of HFD ( n = 10 for Con and n = 6 for MCK-CD47). (M) AMPK phosphorylation and PGC-1α expression in skeletal muscle of MCK-CD47 mice. (N) CS activity in skeletal muscle of MCK-CD47 mice ( n = 11 for Con and n = 7 for MCK-CD47). (O) Schematic of HSA-CD47 mouse model. (P and Q) Growth curves and IPGTT of HSA-CD47 mice on HFD ( n = 8 for Con and n = 6 for HSA-CD47). (R and S) Running distance and duration of HSA-CD47 mice on motor treadmill ( n = 9 for Con and n = 7 for HSA-CD47). (T) CS activity in skeletal muscle of HSA-CD47 mice ( n = 9 for Con and n = 7 for HSA-CD47). (U) AMPK phosphorylation and PGC-1α expression in skeletal muscle of HSA-CD47 mice. (V) Western blot analysis of CD47 and AMPK phosphorylation in skeletal muscle of mice injected with AAV expressing GFP or CD47. Data are presented as means ± SEM and analyzed by two-tailed Student’s t test (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant).

Article Snippet: Milk (5%) in TBST was used to block members for 1 h at room temperature, then the members were washed with TBST three times (5 min each time) and incubated with primary antibodies against P-AMPK (Thr172, 2535), AMPK (2532), P-HSP90α (Thr5/7, 3488), P-ACC (3661), ACC (3676), p-DNA-PK (Ser2056, 68716), COXIV (4850), cyto C (4280), Acetylated-Lysine (9814) (Cell Signaling Technology Inc.), SIRT1 (04-1557, Millipore), PGC-1α (ab54481, Abcam), HSP90α (A5006, ABclonal), DNA-PK (ab70250, Abcam), AMPKγ3 (A14132, ABclonal), AMPKγ1 (A22024, ABclonal), CD47 (AF1866-SP, R&D systems), and GAPDH (AB0037, Abways) in 5% BSA at 4°C overnight.

Techniques: Activation Assay, Knock-Out, Phospho-proteomics, Expressing, Activity Assay, Western Blot, Injection, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: CD47-blocking antibody confers metabolic benefits against obesity

doi: 10.1016/j.xcrm.2025.102089

Figure Lengend Snippet: CD47-blocking antibody promotes AMPK activation by reducing phosphorylation of HSP90α (A) Strategy for analyzing CD47-interacting proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). (B) Endogenous interactions between CD47 and HSP90α detected in myotubes by western blotting. (C and D) HSP90α and DNA-PK phosphorylation in Cd47 knockdown or overexpressing myotubes. (E) DNA-PK inhibitor treatment (10 μM) effects on HSP90α and DNA-PK phosphorylation in Cd47 -overexpressing myotubes. (F) HSP90α and DNA-PK phosphorylation in myotubes treated with CD47-blocking antibody. (G) HSP90α phosphorylation and AMPK activation after treatment with tanespimycin (17-AAG). (H) AMPK phosphorylation in Cd47 -overexpressing myotubes treated with 17-AAG. (I) AMPK phosphorylation after lentiviral treatment with mutated HSP90α phosphorylation sites. (J) The inhibitory effect of Cd47 overexpression on AMPK activation was blocked by mutated HSP90α phosphorylation sites. (K) Schematic: CD47-blocking antibody reduces HSP90α phosphorylation, promoting AMPK activation. Data are presented as means ± SEM and analyzed by two-way RM ANOVA (C, right) and Student’s t test (D, right). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant.

Article Snippet: Milk (5%) in TBST was used to block members for 1 h at room temperature, then the members were washed with TBST three times (5 min each time) and incubated with primary antibodies against P-AMPK (Thr172, 2535), AMPK (2532), P-HSP90α (Thr5/7, 3488), P-ACC (3661), ACC (3676), p-DNA-PK (Ser2056, 68716), COXIV (4850), cyto C (4280), Acetylated-Lysine (9814) (Cell Signaling Technology Inc.), SIRT1 (04-1557, Millipore), PGC-1α (ab54481, Abcam), HSP90α (A5006, ABclonal), DNA-PK (ab70250, Abcam), AMPKγ3 (A14132, ABclonal), AMPKγ1 (A22024, ABclonal), CD47 (AF1866-SP, R&D systems), and GAPDH (AB0037, Abways) in 5% BSA at 4°C overnight.

Techniques: Blocking Assay, Activation Assay, Phospho-proteomics, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Knockdown, Over Expression

Journal: Nature

Article Title: CD47 blocking antibodies restore phagocytosis and prevent atherosclerosis

doi: 10.1038/nature18935

Figure Lengend Snippet: ( a ). Cytoscape network visualization of the genes which are significantly correlated with CD47 expression in both human and murine atherosclerotic plaque reveals a high number of TNF-α-related factors (indicated in blue), including ligands, receptors, and downstream signaling factors. ( b ). PANTHER pathway analysis of those genes which were (a) significantly associated with CD47 expression in mouse and human vascular tissue and (b) have been previously associated with atherosclerosis through the STAGE study , identifies “ inflammation mediated by chemokine and cytokine signaling pathway ” as the most over-abundant pathway associated with CD47 expression in vascular tissue. ( c ). Using the Hybrid Mouse Diversity Panel (HMDP), which correlates aortic gene expression with Luminex cytokine array data of plasma samples from over 100 inbred strains of mice, we found that vascular CD47 expression is positively correlated with three inflammatory cytokines in vivo, including TNF-α, IL-2 and CXCL1. Correlation data shown for CD47 and TNF-α. ( d ). Co-expression studies confirm that TNF-α and CD47 expression are positively correlated in human carotid endarterectomy samples from the BiKE validation study. The Pearson correlation coefficient was determined assuming a Gaussian distribution and P values were determined using a two-tailed test. ( e ). Experiments with primarily cultured mouse aortic SMCs indicate that TNF-α reproducibly induces CD47 mRNA upregulation, while a number of other classical pro-atherosclerotic stimuli have no significant effect. Notably, CXCL1, IL4, TGFβ and IL-2 fail to induce CD47 expression in vitro, as assessed by ANOVA. ( f ). Additional studies suggest that the effect of TNF-α on CD47 expression persists in the presence of oxidized LDL, as occurs in the atherosclerotic plaque. ( g ). Western blotting confirms that TNF-α induces CD47 expression in vascular cells at the protein level. For gel source data, see . ( h ). Immunocytochemistry studies of HCASMCs confirm that CD47 expression is induced on the cell surface of TNF-α treated cells. TNF-α effect is assessed by co-staining for HMGB1, and antibody specificity is confirmed with isotype control and recombinant CD47 peptide quenching assays. ( i ). Multiple assays (including FACS, Taqman and immunocytochemistry studies) reveal that CD47 expression is downregulated on vascular SMCs during programmed cell death, as has previously been observed with inflammatory cells. ( j ). Confirmatory assays in cultured human coronary artery SMC reveal that TNF-α induces changes similar to those observed in murine cells , including an induction of CD47 under physiological conditions and a blunting of its expected downregulation during apoptosis. ( k ). TNF-α’s capacity to impair CD47 downregulation during programmed cell death is also observed in mouse SMCs simultaneously exposed to pro-apoptotic stimuli and oxidized LDL. ( l ). No correlation between CD47 and other candidate cytokines was observed in the BiKE biobank, further supporting a specific relationship between CD47 and TNF-α. ( m ). Representative FACS-based apoptosis panels from cells exposed to the conditions used in confirm that TNF-α suppresses efferocytosis despite increasing programmed cell death. Comparisons made by two-tailed t tests, unless otherwise specified. *** = P < 0.001, * = P < 0.05. Error bars represent the SEM.

Article Snippet: In some experiments, membranes loaded with protein were incubated with anti-CD47 Ab that had been preabsorbed with CD47 peptide (R&D systems, 1866-CD, 1:5 dilution) for 16 hours, to determine the specificity of the primary Ab.

Techniques: Expressing, Gene Expression, Luminex, Clinical Proteomics, In Vivo, Biomarker Discovery, Two Tailed Test, Cell Culture, In Vitro, Western Blot, Immunocytochemistry, Staining, Control, Recombinant

Journal: Nature

Article Title: CD47 blocking antibodies restore phagocytosis and prevent atherosclerosis

doi: 10.1038/nature18935

Figure Lengend Snippet: In vivo serological data and additional in silico and bioinformatic data ( a ). Complete serological studies (including blood count, liver function studies, basic metabolic panel, and fasting glucose) from the 4 week apoE −/− -AngII atherosclerosis model indicate that anti-CD47 Ab induces a significant reduction in hemoglobin and compensatory reticulocytosis, consistent with prior reports 4 , 7 . The erythrophagocytosis of senescent RBCs appears to be self-limited, and no anemia was observed in the chronic atherosclerosis model or the reduced dose model (P = 0.54 and 0.57, respectively). No significant difference in any other serum marker is observed except for an increase in serum creatinine, which does not deviate outside of the reference range. Metabolic parameters and leukocyte differential data from the 12 week chronic atherosclerosis model are displayed at the bottom of the table. ( b ). Additional Upstream Regulator Analysis (URA) bioinformatic analyses of the Cytoscape data displayed in Extended Data Figure 7a performed within the Ingenuity Pathway Analysis (IPA) software identifies a number of TNF-α related factors (indicated in red) which are predicted to mediate transcriptional regulatory roles in the gene network shown in that panel. P-values were determined from Fisher’s Exact Test by comparing overlap of co-expressed genes with known upstream regulators from the Ingenuity Knowledge Base. ( c ). Several additional DAVID -based bioinformatics analyses including ( KEGG , SMART , PANTHER and GO analyses) confirm the association between CD47 and inflammatory signaling related to the TNF-α pathway (indicated in red). Blue panels indicate the –logp10 value for each identified factor. ( d ). Transcription factor binding site prediction algorithms identify several putative NFKB family binding sites within the CD47 promoter, as displayed in Extended Data Figure 8a . ( d ). List of primers used in this study.

Article Snippet: In some experiments, membranes loaded with protein were incubated with anti-CD47 Ab that had been preabsorbed with CD47 peptide (R&D systems, 1866-CD, 1:5 dilution) for 16 hours, to determine the specificity of the primary Ab.

Techniques: In Vivo, In Silico, Marker, Software, Binding Assay

Journal: bioRxiv

Article Title: EV trace : tracing extracellular vesicles-associated proteins in recipient cells using stable isotope labeling

doi: 10.1101/2025.11.23.690082

Figure Lengend Snippet: ( A ) Distribution of the amount of clusters (indicative of EVs) per size range (nm, depicting the diameter of the clusters) detected using the different staining panels (black circle, Integrin-β1+WGA; blue square, Integrin-β1+GFRP78; red triangle, CD47+GRP78). ( B ) Representative images of individual EVs that were enriched in either one (left, right) or two (middle) of the indicated markers: WGA (green), integrin-β1 (magenta), GRP78 (cyan), CD47 (yellow). Each horizontal row represents one of the three staining panels. White scale bars indicate 200 nm. ( C ) The percentage of EVs (of all clusters detected in each staining panel as in A) that are single or double positive for indicated markers. Bars are colored to indicate markers: light grey, WGA; blue horizontal stripes, integrin-β1; dark grey, GRP78; red vertical stripes, CD47. ( D ) Graphical summary of the detected PCa EV subpopulations and in which cell types these markers were traced (see ). Colors indicating the proteins are similar as in C. A and C show mean±SEM of 3 fields of view. WGA, Wheat Germ Agglutinin

Article Snippet: Unconjugated integrin-β1 antibody (clone P5D2, Santa Cruz Biotechnology, Dallas, TX, USA), CD47 antibody (clone B6.H12, InVivo MAb), or mouse IgG1 isotype control (clone P3.6.2.8.1, eBioscience, Invitrogen) were labeled with mouse IgG1 targeting nanobodies (VHH-AlexaFluor568, VHH-AlexaFluor647, Proteintech, Rosemont, IL, USA).

Techniques: Staining