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: 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: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis

doi: 10.1161/ATVBAHA.122.318904

Figure Lengend Snippet: CD47 (cluster of differentiation) mediates TSP1 (thrombospondin-1)-induced inhibition of lymphangiogenesis. A , Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the relative mRNA levels of CD47 and CD36 in human lymphatic endothelial cells (HLECs) and human aortic endothelial cells (HAoEC). Bar graph represents mRNA levels in comparison to CD36 (n=9). B , Control and CD47 siRNA-treated lymphatic endothelial cells (LECs; 48 h) were utilized to quantify CD47 transcript expression using qRT-PCR (n=6). C , WST (water-soluble tetrazolium)-1 assay was conducted to investigate the effects of CD47 silencing on LEC proliferation in response to TSP1 treatment as described in Figure 2A. Data are representative of 3 independent experiments performed at least in quadruplicate. D , Control and CD47 -silenced LECs were used to evaluate cell migration. Scale bar, 200 µm. xBar graph represents the percentage of migrated cells (n=3–4). E through G , Control and CD47 -silenced cells were pretreated as in Figure 2D and seeded in wells of a Matrigel-coated plate in basal medium containing VEGF (vascular endothelial growth factor)-C±TSP1 and tube formation determined. Representative images of tube formation are shown ( E ). Scale bar, 1000 µm. Tube length ( F ) and number of branching points ( G ) quantified (n=7). H , Wild-type male mice were injected SC with Matrigel solutions premixed with either VEGF-C, VEGF-C+TSP1+IgG, or VEGF-C+TSP1+CD47-blocking antibody. Plugs were isolated after 10 days, sectioned, and immunostained for LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1). Representative images of LYVE-1 staining of the cross-sections of the Matrigel plugs and quantification of LYVE-1–positive area are shown (n=5–7). Scale bar, 20 μm. Statistical analyses were performed using 2-way ANOVA ( A , C , D , F , and G ) with Bonferroni ( A ), Tukey ( C , F , and G ), and Sidak ( D ) multiple comparisons test, 2-tailed unpaired Student t test ( B ), and Kruskal-Wallis test for multiple comparisons ( H ). Data represent mean±SEM.

Article Snippet: CD47 antibody (NBP2-31106; Novus Biologicals, LLC, Centennial, CO) and ab175388 (Abcam, Cambridge, MA) were used.

Techniques: Inhibition, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Comparison, Control, Expressing, Migration, Injection, Blocking Assay, Isolation, Staining

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis

doi: 10.1161/ATVBAHA.122.318904

Figure Lengend Snippet: TSP1 (thrombospondin-1)-induced CD47 (cluster of differentiation) activation blocks VEGF (vascular endothelial growth factor)-C–stimulated lymphangiogenic signaling. A through D , Lymphatic endothelial cells (LECs) were pretreated with TSP1 (22 nM, 16 h) in 0.5% fetal bovine serum (FBS) containing basal media MV2, stimulated with VEGF-C (15 min), and cell lysates subjected to Western blot analysis. A , Representative Western blot images are shown. B through D , Bar diagrams represent mean protein levels expressed as a ratio of phospho-to-total proteins, AKT ( B ), eNOS ( C ), and ERK1/2 ( D ; n=3). E , LECs were treated with control or CD47 -siRNA (48 h) and immunoblotting done to determine CD47 expression (ab175388). F through I , Control or CD47 -siRNA–treated cells were treated as in Figure 4A and Western blot experiments executed. F , Representative Western blot images are shown. G through I , Bar diagrams represent pAKT/total AKT ( G ), peNOS/total eNOS ( H ), and pERK1/2/total ERK1/2 ( I ; n=3). J , Control or CD47 -silenced LECs were pretreated with TSP1 (22 nM, 4 h), stimulated with VEGF-C (100 ng/mL, 1 h) and analyzed for NO (nitric oxide) production using DAF-FM diacetate. K , Control or CD47 -silenced LECs were pretreated with vehicle or TSP1 (1 h), incubated with H2DCFDA solution, and fluorescence analyzed using flow cytometry. Statistical analyses were performed using 1-way ANOVA ( B–D ), 2-way ANOVA with Tukey test for multiple comparisons ( G–I and K ), and 2-tailed unpaired Student t test ( J ). Data represent mean±SEM.

Article Snippet: CD47 antibody (NBP2-31106; Novus Biologicals, LLC, Centennial, CO) and ab175388 (Abcam, Cambridge, MA) were used.

Techniques: Activation Assay, Western Blot, Control, Expressing, Incubation, Fluorescence, Flow Cytometry

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis

doi: 10.1161/ATVBAHA.122.318904

Figure Lengend Snippet: Lymphatic endothelial cell (LEC)–specific Cd47 deficiency reduces atherosclerotic lesion formation. A through M , Male Cd47 WT and Cd47 ΔLEC mice were injected with AAV (adeno-associated virus) 8- PCSK9 IP, fed a Western diet for 16 weeks and atherosclerosis analyzed. A , Representative in situ images of aortic arch (yellow arrowheads point to atherosclerotic lesions). Scale bar, 2 mm. B , Representative en face oil red O (ORO) staining of aorta. Scale bar, 5 mm. C , Quantification of plaque area in aorta (n=13–14). D through G , Bar diagrams show plasma total cholesterol ( D ), plasma triglycerides ( E ), fasting blood glucose levels ( F ), and body composition (fat, lean, and fluid mass; G ; n=8–13). H , Representative images of aortic root cross-sections stained with H&E (neointima area), ORO (lipid accumulation), Masson trichrome (collagen content), CD68 (cluster of differentiation; macrophage burden), and necrotic area (encircled in red). Scale bar, 200 µm. I through M , Bar diagrams show neointima area ( I ), lipid deposition ( J ), collagen content ( K ), macrophage accumulation ( L ), and necrotic area ( M ) in aortic root sections (n=5–9). Statistical analyses were performed using a 2-tailed unpaired Mann-Whitney U test ( C , E , and M ), 2-tailed unpaired Student t test ( D , F , and I–L ), and 2-way ANOVA followed by Sidak post hoc test ( G ). Data represent mean±SEM.

Article Snippet: CD47 antibody (NBP2-31106; Novus Biologicals, LLC, Centennial, CO) and ab175388 (Abcam, Cambridge, MA) were used.

Techniques: Injection, Virus, Western Blot, In Situ, Staining, Clinical Proteomics, MANN-WHITNEY

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis

doi: 10.1161/ATVBAHA.122.318904

Figure Lengend Snippet: Lymphatic endothelial cell (LEC)–specific deletion of Cd47 (cluster of differentiation) in mice increases arterial lymphatic vessel (LV) density. A through D , Aortic root cross-sections from male AAV (adeno-associated virus) 8- PCSK9 –injected Cd47 WT and Cd47 ΔLEC mice (16-wk Western diet) were immunostained for CD68, iNOS (inducible NO [nitric oxide] synthase), Arg1 (arginase 1), and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1). Nuclei were counterstained with DAPI (4’,6-diamidino-2-phenylindole; blue). Representative confocal images of iNOS (green; A ) and CD68 (red); Arg1 (green; B ) and CD68 (red; scale bar, 50 µm); and LYVE-1 staining (red; C ; scale bar, 20 µm) are shown (n=5–6). Statistical analyses were performed using a 2-tailed unpaired Student t test ( A and B ) and a Mann-Whitney U test ( C ). Data represent mean±SEM. A indicates adventitia; M, media; and P, plaque.

Article Snippet: CD47 antibody (NBP2-31106; Novus Biologicals, LLC, Centennial, CO) and ab175388 (Abcam, Cambridge, MA) were used.

Techniques: Virus, Injection, Western Blot, Staining, MANN-WHITNEY

Journal: Oncoimmunology

Article Title: Bendamustine with total body irradiation conditioning yields tolerant T-cells while preserving T-cell-dependent graft-versus-leukemia

doi: 10.1080/2162402X.2020.1758011

Figure Lengend Snippet: BEN-TBI does not result in appreciable donor T-cell phenotypic differences post-transplant when compared to CY-TBI. (a–c) BALB/c recipient mice received 40 mg/kg BEN iv or 200 mg/kg CY ip on day −2, 400 cGy TBI on day −1, and 10 7 TCD-BM from naïve C57BL/6 mice with 3 × 10 6 CellTrace Violet-stained tT from naïve BoyJ mice on day 0. Blood and spleen were collected on day +3. (a) % donor T-cells (CD45.1+) was determined by flow cytometry. Using CBCs determined by HemaVet analysis, absolute number of donor T-cells was calculated. (b) After gating on CD45.1+ cells (representing donor T-cells), CellTrace Violet dilution was analyzed using ModFit software to determine proliferation index. Representative CellTrace Violet dilution is shown. (c) Within the CD45.1+ gate, cells were stratified by CellTrace high (non- proliferative) and CellTrace low (proliferative) and CD25 and CD47 expression were analyzed by flow cytometry. Pooled data from 2 experiments with line at mean are shown, n = 6–7 mice/group. * p < .05 , ** p < .01 . (d-g) BALB/c recipient mice received 40 mg/kg BEN iv or 200 mg/kg CY ip on day −2, 400 cGy TBI on day −1, and 10 7 BM with 3 × 10 6 SC from naïve C57BL/6 mice on day 0. Peripheral blood was collected on days +7, +14, +21, +35, and +70 and stained for CD8, CD4, Tbet, GATA3, RORγt, CD134, CD278, PD-1, TIM3, CTLA-4, and CD272. CBCs were determined and used to calculate absolute cell numbers. Average absolute numbers of cells per μL of blood are shown with SEM. Representative flow plots from day +7 with fluorescence minus one (FMO) controls are shown (e). (d) Pooled data from 4 experiments are shown, n = 19 mice/group. (e-g) Pooled data from 2 experiments are shown, n = 10 mice/group. ** p < .01.

Article Snippet: Antibodies used were anti-mouse H2kb PerCP-eFluor710 (clone AF6-88.5.5.3; 46–5958), CD8α PE-Cy7 (53–6.7; 25–0081), CD4 APC (RM4-5; 50-148-54), FoxP3 APC (FJK-16s; 17–5773), CD45.1 APC (A20; 17–0453), GATA3 PE-Cy7 (TWAJ; 25–9966), CD134 PE-Cy7 (OX-86; 25–1341), FoxP3 PE (150D/E4; 12–4774), CD69 PE-Cy5 (H1.2F3; 15–0691), RORγt APC (AFKJS-9; 17–6988), IFN gamma PE (XMG1.2, 12–7311) (Thermo Fisher Scientific), CD45.1 PE-CF594 (A20; 562452), CD3ε PE-CF594 (145–2 C11; 562286), CD44 BB515 (IM7; 565941), CD44 BV510 (IM7; 563114) (BD Biosciences), CD47 PE-Vio770 (REA170; 130-102-383), TIM-3 VioBright FITC (REA602; 130-109-449), TIM-3 PE (REA602; 130-118-563), CD278 VioGreen (REA192; 130-100-739), CD272 PE (REA224; 130-102-689), CD4 APC-Vio770 (GK1.5; 130-102-786), CD4 VioGreen (GK1.5; 130-102-444) (Miltenyi Biotec), Tbet PE-Dazzle594 (4B10; 644828), CTLA-4 PE-Dazzle594 (UC10-4B9; 106318), CD8α Brilliant Violet 421 (53–6.7; 100738), CCR7 PE-Cy5 (4B12; 120114), PD-1 APC (29F.1A12; 135210), CD25 AlexaFluor700 (PC61; 102024), and TNF-α Brilliant Violet 510 (MP6-XT22, 506339) (Biolegend).

Techniques: Staining, Flow Cytometry, Software, Expressing, Fluorescence

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: EGFR-Induced and c-Src-Mediated CD47 Phosphorylation Inhibits TRIM21-Dependent Polyubiquitylation and Degradation of CD47 to Promote Tumor Immune Evasion.

doi: 10.1002/advs.202206380

Figure Lengend Snippet: Figure 1. CD47 is highly expressed in GBM and correlates with a poor prognosis in GBM patients. A) Relative mRNA expression levels of the indicated genes were analyzed in the TCGA cohort of GBM (n = 162). B) Kaplan–Meier 18-year overall survival analysis comparing CD47 high- and low-expressing patients in the TCGA GBM cohort. The CD47 high and low groups were separated by the median expression. Significance was determined with the log- rank test. p = 5.6 × 10−16; HR, 2.8. C) IHC staining of 75 human glioma specimens of different grades (II–IV) was performed with an anti-CD47 antibody. Representative images of IHC staining from the specimens are shown. Scale bar, 100 μm. D) Immunoblotting analysis of CD47 protein expression in paired tumor-adjacent normal tissues (N) and human GBM specimens (T). E) The protein expression levels of CD47 in NHA cells and the indicated human GBM and glioma stem cells (GSCs) were determined by immunoblotting analyses.

Article Snippet: Polyclonal antibodies against human CD47 (AF4670, 1 μg mL−1 for immunoblotting) and mouse CD47 (AF1866, 1 μg mL−1 for immunoblotting) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Immunohistochemistry, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: EGFR-Induced and c-Src-Mediated CD47 Phosphorylation Inhibits TRIM21-Dependent Polyubiquitylation and Degradation of CD47 to Promote Tumor Immune Evasion.

doi: 10.1002/advs.202206380

Figure Lengend Snippet: Figure 3. c-Src activation induces CD47 protein upregulation in response to EGFR activation. Immunoblot analyses were performed with the indicated antibodies (A–E). A) Serum-starved U251 cells were pretreated with the indicated inhibitors for 2 h and then stimulated with EGF (100 ng mL−1) for 24 h. B) Serum-starved U251 cells with stable expression of different shRNAs against c-Src or a control shRNA were stimulated with or without EGF (100 ng mL−1) for 24 h. C) U251 cells were transfected with a control vector, wild-type (WT) c-Src, or an active c-Src (CA) for 48 h. D) U87/EGFRvIII cells were pretreated with DMSO or Su6656 (4 μm) for 2 h and then treated with CHX (100 μg mL−1) for the indicated periods of time. Quantification of relative CD47 protein levels is shown (bottom panel). E) U251 cells stably expressed c-Src WT or c-Src CA were treated with CHX (100 μg mL−1) for the indicated periods of time. Quantification of relative CD47 protein levels is shown (right panel).

Article Snippet: Polyclonal antibodies against human CD47 (AF4670, 1 μg mL−1 for immunoblotting) and mouse CD47 (AF1866, 1 μg mL−1 for immunoblotting) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Western Blot, Expressing, Control, shRNA, Transfection, Plasmid Preparation, Stable Transfection

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: EGFR-Induced and c-Src-Mediated CD47 Phosphorylation Inhibits TRIM21-Dependent Polyubiquitylation and Degradation of CD47 to Promote Tumor Immune Evasion.

doi: 10.1002/advs.202206380

Figure Lengend Snippet: Figure 4. c-Src binds to and phosphorylates CD47 at Y288, which subsequently upregulates CD47 stability by inhibiting CD47 polyubiquitylation. Im- munoblotting analyses were performed with the indicated antibodies (A–K). A) Serum-starved U251 cells were stimulated with or without EGF (100 ng

Article Snippet: Polyclonal antibodies against human CD47 (AF4670, 1 μg mL−1 for immunoblotting) and mouse CD47 (AF1866, 1 μg mL−1 for immunoblotting) were obtained from R&D Systems (Minneapolis, MN).

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: EGFR-Induced and c-Src-Mediated CD47 Phosphorylation Inhibits TRIM21-Dependent Polyubiquitylation and Degradation of CD47 to Promote Tumor Immune Evasion.

doi: 10.1002/advs.202206380

Figure Lengend Snippet: Figure 5. CD47 Y288 phosphorylation inhibits TRIM21-mediated CD47 K99/102 polyubiquitylation and CD47 degradation. Immunoblotting analyses were performed with the indicated antibodies (A–J). A) U251 cells were treated with or without EGF (100 ng mL−1) for 1 h. Immunoprecipitation was

Article Snippet: Polyclonal antibodies against human CD47 (AF4670, 1 μg mL−1 for immunoblotting) and mouse CD47 (AF1866, 1 μg mL−1 for immunoblotting) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: EGFR-Induced and c-Src-Mediated CD47 Phosphorylation Inhibits TRIM21-Dependent Polyubiquitylation and Degradation of CD47 to Promote Tumor Immune Evasion.

doi: 10.1002/advs.202206380

Figure Lengend Snippet: Figure 7. Activation of the EGFR/c-Src pathway correlates with CD47 expression in human GBM specimens. A) IHC staining of 25 human GBM speci- mens was performed with the indicated antibodies. Representative images from the staining of seven different specimens are shown. Scale bar, 100 μm. B,C) The IHC stains were scored, and correlation analyses were performed. The Pearson correlation test was used. Note that the scores of some samples overlap. D) A schematic of c-Src-regulated CD47 phosphorylation and expression.

Article Snippet: Polyclonal antibodies against human CD47 (AF4670, 1 μg mL−1 for immunoblotting) and mouse CD47 (AF1866, 1 μg mL−1 for immunoblotting) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Expressing, Immunohistochemistry, Staining, Phospho-proteomics

Journal: Frontiers in Oncology

Article Title: YB-1-based oncolytic virotherapy in combination with CD47 blockade enhances phagocytosis of pediatric sarcoma cells

doi: 10.3389/fonc.2024.1304374

Figure Lengend Snippet: Infection with XVir-N-31 (XVir) increases both calreticulin (CALR) and CD47 surface expression on pediatric sarcoma cell lines. (A) Analysis of CALR (‘eat-me’) and CD47 surface expression (‘don’t-eat-me’) surface expression of pediatric sarcoma cell lines A673, SKNMC, and U2OS 48 hours post infection (hpi) at indicated multiplicity of infection (MOI) assessed by FACs analysis after dead cell exclusion via DAPI. Y-axis depicts the fold change of expression compared to controls (ctrl) using frequency of parent minus isotype (IT). (B) Analysis MOI/dose-dependency of CD47 surface expression at 48hpi using indicated MOI. Statistical analysis was performed using the unpaired student’s t-test in (A) and one way ANOVA with multiple comparison and Tukey correction in (B) . Plotted is the mean with SD. Each dot represents one biological replicate. Experiments were repeated at least twice to ensure reproducibility. Levels of significance are indicated as asterisks *p<0,0332; **p<0,0021; ***p<0,0002; ****p<0,0001.

Article Snippet: Following antibodies were used: Human CD47 Antibody, (#AF4670, R&D Systems, 1:500), Adenovirus-Hexon (#AB1056, Merck Millipore, 1:1000), GAPDH (#2118S, Cell Signaling Technology, 1:2000), Mouse anti-rabbit IgG HRP (#Sc-2357, Santa Cruz Biotech, 1:1000), anti-sheep IgG HRP (#HAF016, R&D Systems, 1:1000).

Techniques: Infection, Expressing, Comparison

Journal: Frontiers in Oncology

Article Title: YB-1-based oncolytic virotherapy in combination with CD47 blockade enhances phagocytosis of pediatric sarcoma cells

doi: 10.3389/fonc.2024.1304374

Figure Lengend Snippet: The combination (combo) of XVir-N-31 (XVir) and the CD47-inhbitor (CD47i) B6H12.2 shows the highest levels of phagocytosis for all tested phagocytes and cell lines. Phagocytosis by THP-1 macrophages (A) , THP-1 imDCs (B) , and healthy donor-derived monocytic imDCs (C) was assessed at indicated MOI (48hpi) and time (y-axis) for A673 (left panels) and U2OS (right panels). CD47i was added when starting phagocytosis. Each dot represents one biological replicate. Experiments were repeated at least three times. Plotted is the normalized phagocytosis compared to ctrl as mean and SD. One way ANOVA with multiple comparison and Tukey correction was used for statistical analysis. Levels of significance are indicated as asterisks *p<0,0332; **p<0,0021; ***p<0,0002; ****p<0,0001.

Article Snippet: Following antibodies were used: Human CD47 Antibody, (#AF4670, R&D Systems, 1:500), Adenovirus-Hexon (#AB1056, Merck Millipore, 1:1000), GAPDH (#2118S, Cell Signaling Technology, 1:2000), Mouse anti-rabbit IgG HRP (#Sc-2357, Santa Cruz Biotech, 1:1000), anti-sheep IgG HRP (#HAF016, R&D Systems, 1:1000).

Techniques: Derivative Assay, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: Metformin mediates induction of miR‐708 to inhibit self‐renewal and chemoresistance of breast cancer stem cells through targeting CD47

doi: 10.1111/jcmm.14462

Figure Lengend Snippet: Clinicopathological variables and miR‐708 expression in 473 breast cancer patients

Article Snippet: Cell lysates were prepared and the corresponding proteins were separated by SDS/PAGE, and probed with primary antibodies against CD47 (Novus Biologicals) and then HRP‐conjugated secondary antibodies (GE Healthcare) were used, with the anti‐actin antibody as an internel control (Santa Cruz).

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Metformin mediates induction of miR‐708 to inhibit self‐renewal and chemoresistance of breast cancer stem cells through targeting CD47

doi: 10.1111/jcmm.14462

Figure Lengend Snippet: CD47 was a direct target of miR‐708 in breast cancer. A, Schematic of putative miR‐708 binding sequence in the 3′‐UTR of CD47, and the generation of mutation of CD47. B and C, Relative luciferase activity of CD47 wt or mut 3′‐UTR was detected in MDA‐MB‐231.SC and MCF‐7.SC transfected with miR‐708. D and E, CD47 expression was detected in the miR‐708 interfered MDA‐MB‐231.SC and MCF‐7.SC by real‐time PCR (D) and western blot (E). NC, negative control. ** P < 0.01

Article Snippet: Cell lysates were prepared and the corresponding proteins were separated by SDS/PAGE, and probed with primary antibodies against CD47 (Novus Biologicals) and then HRP‐conjugated secondary antibodies (GE Healthcare) were used, with the anti‐actin antibody as an internel control (Santa Cruz).

Techniques: Binding Assay, Sequencing, Mutagenesis, Luciferase, Activity Assay, Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Negative Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Metformin mediates induction of miR‐708 to inhibit self‐renewal and chemoresistance of breast cancer stem cells through targeting CD47

doi: 10.1111/jcmm.14462

Figure Lengend Snippet: Knockdown of CD47 expression eradicates the BCSCs. A, MDA‐MB‐231 and MCF‐7 cells transfected with shCD47 formed smaller mammospheres than cells transfected with NC, and the number of formed mammospheres were counted in 5 random fields on day 14. For mammosphere formation assay, 3000 cells were used, and mammospheres were counted with size >100 mm under microscope. B, Flow cytometry analyses of the CD44 + /CD24 − population in MDA‐MB‐231 and MCF‐7 cells transfected with shCD47 in mammosphere culture conditions using fluorescentconjugated CD44 and CD24 antibodies. The data represent the mean of 3 independent experiments. C, The numbers of animals with detectable tumours in the groups injected with the MDA‐MB‐231 mammosphere cells (with or without shCD47 transfection before), were inoculated into the mammary fat pad of BALB/C nude mice. D, The detectable tumours in mice were measured for tumour weight 1 month later. E and F, Kaplan‐Meier curves for OS (E) and DFS (F) of breast cancer patients with low vs high expression of CD47 in SYSUCC. A total of 473 patients with breast cancer were divided into low CD47 (n = 241) and high CD47 groups (n = 232). NC, negative control. * P < 0.05, ** P < 0.01

Article Snippet: Cell lysates were prepared and the corresponding proteins were separated by SDS/PAGE, and probed with primary antibodies against CD47 (Novus Biologicals) and then HRP‐conjugated secondary antibodies (GE Healthcare) were used, with the anti‐actin antibody as an internel control (Santa Cruz).

Techniques: Knockdown, Expressing, Transfection, Tube Formation Assay, Microscopy, Flow Cytometry, Injection, Negative Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Metformin mediates induction of miR‐708 to inhibit self‐renewal and chemoresistance of breast cancer stem cells through targeting CD47

doi: 10.1111/jcmm.14462

Figure Lengend Snippet: CD47 deficiency or miR‐708 overexpression can increase the phagocytosis and chemosensitivity of breast cancer cells. A, The MDA‐MB‐231, MDA‐MB‐231.SC, MCF‐7 and MCF‐7.SC cells were transfected to NC, shCD47 and miR‐708 for 48 h, incubated with bone marrow‐derived macrophages for 2 h, and the percentage of phagocytosed cancer cells was determined and normalized to the NC at adherent cells. B, Cell viability was measured through CCK‐8 assay to evaluate the chemosensitivity of shCD47 and anti‐miR‐708 in MDA‐MB‐231. NC, negative control. * P < 0.05, ** P < 0.01

Article Snippet: Cell lysates were prepared and the corresponding proteins were separated by SDS/PAGE, and probed with primary antibodies against CD47 (Novus Biologicals) and then HRP‐conjugated secondary antibodies (GE Healthcare) were used, with the anti‐actin antibody as an internel control (Santa Cruz).

Techniques: Over Expression, Transfection, Incubation, Derivative Assay, CCK-8 Assay, Negative Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Metformin mediates induction of miR‐708 to inhibit self‐renewal and chemoresistance of breast cancer stem cells through targeting CD47

doi: 10.1111/jcmm.14462

Figure Lengend Snippet: Metformin induces miR‐708‐mediated suppression of CD47. A and B, Metformin increased the miR‐708 (A) and decreased the CD47 (B) expression level in MDA‐MB‐231.SC and MCF‐7.SC. The cells were treated with 10 mM metformin or PBS for 48 h and the mRNA levels were determined by quantitative RT‐PCR. The statistical significance was determined by student's t test. C and D, Immunoblot analyses of CD47 expression in MDA‐MB‐231.SC anti‐miR‐708 (C) and MCF‐7.SC anti‐miR‐708 (D) cells incubated with metformin (0.3‐3.0 mM) for 72 h. β‐actin was used as a loading control. E, A schematic model of the mechanism underlying the role of metformin and miR‐708 in BCSCs self‐renewal and chemoresistance. * P < 0.05, ** P < 0.01

Article Snippet: Cell lysates were prepared and the corresponding proteins were separated by SDS/PAGE, and probed with primary antibodies against CD47 (Novus Biologicals) and then HRP‐conjugated secondary antibodies (GE Healthcare) were used, with the anti‐actin antibody as an internel control (Santa Cruz).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Incubation, Control