sku human cd47 skov3 ovcar8  (OriGene)

Journal: Molecular therapy oncolytics

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

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 1. CD47 levels in human lung adenocarcinoma cells were increased following tumor metastasis (A and B) Comparison of CD47 expression between primary lung adenocarcinoma and lung adenocarcinoma metastases in the lymph nodes. (C and D) Comparison of CD47 expression between primary lung adenocarcinoma and lung adenocarcinoma metastases in the liver. (A) and (C) showed representative IHC images for CD47 staining from 14 to 5 lung adenocarcinoma tissue samples, respectively. Data were presented as means ± SDs. **p < 0.01. Scale bar, 50 mm.

Article Snippet: For the reporter analyses, CD47 promoter WT plasmid containing the IRF-1 binding site was generated by inserting the fragment (600 bp, obtained from the University of California, Santa Cruz [UCSC] database, http://genome-asia.ucsc.edu), from A549 genomic DNA into the multicloning site (MCS) of the PGL3 Basic Vector (Promega, Madison, WI, USA).

Techniques: Comparison, Expressing, Staining

Journal: Molecular therapy oncolytics

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

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 2. Induction of CD47 by IFN-g in human lung cancer cells (A) Flow cytometry analysis of CD47 surface expression in human lung cancer cell lines upon incubation with recombinant IFN-g (100 ng/mL, 24 h). Right: Representative image; left: quantitative analysis. (B) Immunofluorescence analysis of CD47 expression in human lung cancer cell lines upon incubation with recombinant IFN-g (100 ng/mL, 24 h). Scale bar, 20 mm. (C) Western blot analysis of CD47 expression in human lung cancer cell lines after IFN-g treatment. (D) qRT-PCR analysis of CD47 mRNA in human lung cancer cell lines upon incubation with recombinant IFN-g (100 ng/mL, 24 h). Left: Representative image; right: quantitative analysis. Data from more than 3 independent experiments were presented as means ± SDs. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: For the reporter analyses, CD47 promoter WT plasmid containing the IRF-1 binding site was generated by inserting the fragment (600 bp, obtained from the University of California, Santa Cruz [UCSC] database, http://genome-asia.ucsc.edu), from A549 genomic DNA into the multicloning site (MCS) of the PGL3 Basic Vector (Promega, Madison, WI, USA).

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

Journal: Molecular therapy oncolytics

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

doi: 10.1016/j.omto.2022.04.011

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

Article Snippet: For the reporter analyses, CD47 promoter WT plasmid containing the IRF-1 binding site was generated by inserting the fragment (600 bp, obtained from the University of California, Santa Cruz [UCSC] database, http://genome-asia.ucsc.edu), from A549 genomic DNA into the multicloning site (MCS) of the PGL3 Basic Vector (Promega, Madison, WI, USA).

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

Journal: Molecular therapy oncolytics

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

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 4. IFN-g induces CD47 expression in lung cancer cells through IRF-1 (A) Sequence of the CD47 promoter showing the position of the putative IRF-1-binding site. (B) Reporter assay for putative IRF-1 binding. The results were normalized to relative luciferase units (RLUs). (C) ChIP assay analyzing the CD47 promoter in A549 cells. The results were normalized to the input. (D and E) IRF-1 mRNA (D) and protein (E) levels in A549 cells transfected with IRF-1-specific shRNA or scramble shRNA after IFN-g treatment. (F and G) CD47 level in A549 cells transfected with IRF-1-specific shRNA or scramble shRNA after IFN-g treatment detected by flow cytometry (F) and immunofluorescence (G). Scale bar, 10 mm. (H) Western blot analysis of CD47 levels in A549 cells transfected with IRF-1-specific or scramble shRNA. Left: Representative image; right: quantitative analysis. Data from 3 independent experiments were presented as means ± SDs. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: For the reporter analyses, CD47 promoter WT plasmid containing the IRF-1 binding site was generated by inserting the fragment (600 bp, obtained from the University of California, Santa Cruz [UCSC] database, http://genome-asia.ucsc.edu), from A549 genomic DNA into the multicloning site (MCS) of the PGL3 Basic Vector (Promega, Madison, WI, USA).

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

Journal: Molecular therapy oncolytics

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

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 5. IFN-g promotes lung cancer cell metastasis by upregulating CD47 expression in vitro (A) Scratch-wound healing assay in A549 cells (A549-WT) and CD47-knockout A549 cells (A549-CD47-KO) were treated with/without IFN-g. (B) Transwell assay in A549 cells (A549-WT) and A549-CD47-KO cells were treated with/without IFN-g. Data from 3 independent experiments were presented as means ± SDs. **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar, 200 mm.

Article Snippet: For the reporter analyses, CD47 promoter WT plasmid containing the IRF-1 binding site was generated by inserting the fragment (600 bp, obtained from the University of California, Santa Cruz [UCSC] database, http://genome-asia.ucsc.edu), from A549 genomic DNA into the multicloning site (MCS) of the PGL3 Basic Vector (Promega, Madison, WI, USA).

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

Journal: Molecular therapy oncolytics

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

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 6. IFN-g promotes human lung cancer cell metastasis in immunodeficient mice by upregulating CD47 (A) A549 cells (A549-WT) and A549-CD47-KO were engrafted in the lungs of the BALB/c-nude mice. Three weeks post-engraftment, the mice were randomly divided into 2 groups. One group was administered with IFN-g (10 ng/mouse, injected once every 2 days) (A549-WT + IFN-g; A549-CD47-KO + IFN-g), and the other group without IFN-g injection was served as control (A549-WT; A549-CD47-KO). After 5 weeks, the mice were sacrificed to analyze tumor growth and metastasis. (B and C) H&E staining and human CD47 immune staining in mouse lungs (B) and livers (C). In both (B) and (C), left: representative images; right: quantification of tumor nodules. Data were presented as means ± SDs. *p < 0.05, ***p < 0.001. NS, no significance.

Article Snippet: For the reporter analyses, CD47 promoter WT plasmid containing the IRF-1 binding site was generated by inserting the fragment (600 bp, obtained from the University of California, Santa Cruz [UCSC] database, http://genome-asia.ucsc.edu), from A549 genomic DNA into the multicloning site (MCS) of the PGL3 Basic Vector (Promega, Madison, WI, USA).

Techniques: Injection, Control, Staining

Journal: Journal of Cell Science

Article Title: Macrophages show higher levels of engulfment after disruption of cis interactions between CD47 and the checkpoint receptor SIRPα

doi: 10.1242/jcs.237800

Figure Lengend Snippet: Treatment of macrophages with anti-CD47 increases phagocytosis. (A) (i) Recent trials of anti-CD47 therapies have shown efficacy only when combined with anti-cancer opsonizing antibodies. In particular, a primary mechanism of anti-CD20-mediated clearance of B-cell lymphoma is via IgG-activated phagocytosis by liver macrophages (i.e. Kupffer cells), requiring B-cells to enter the blood circulation (Montalvao et al., 2013). (ii) Blockade of CD47 often causes loss of blood cells, especially RBCs, presumably through splenic macrophages. (B) Bar graph, CD47 and SIRPα expression on diverse human-derived cell types, including hematopoietic cells (stem cells, HSCs; PBMC, peripheral blood monocytic cells, a THP-1 monocyte line and RBCs), plus mesenchymal stem cells (MSCs), and A549 lung adenocarcinoma cells. HSCs were differentiated to myeloid cells by treatment with G-CSF according to Shin et al. (2013). Quantification by flow cytometry used primary antibodies (B6H12, SEC72 clones) labeled with fluorescein (mean±s.e.m. for all results; n=4). Table, molecular density estimates for CD47 and SIRPα on several hematopoietic cell types based on Subramanian et al. (2007). (C) Phagocytosis assays used THP-1 macrophages (Mφ) and human RBC (hRBC) opsonizing antibody with or without anti-CD47 (B6H12) at saturating level. Engulfment of fluorescent hRBCs was confirmed with a secondary antibody against anti-hRBC. Microscopy fields are randomly selected, and >200 macrophages counted (n=3, mean±s.d.). Hyperbolic fits y=A x/(K+x) of each data set [(A,K): (74%, 67 nM) for anti-CD47, (63%, 125 nM) for control; R2>0.97] give the (inset) difference curve with the difference between A values as Δsat. Scale bar: 20 μm. (D) Anti-CD47 pre-incubated for 45 min with THP-1 macrophages (and excess removed) increased phagocytosis of target hRBCs, independently of hRBC opsonization. Results are mean±s.d., n=3. *P<0.03, n.s., not significant.

Article Snippet: shRNA lentiviral supernatants to CD47 were purchased from Millipore Sigma (TRC#: TRCN0000007836, TRCN0000007837) to target CD47 and resulted in 52 and 87% knockdown, respectively.

Techniques: Expressing, Derivative Assay, Flow Cytometry, Clone Assay, Labeling, Microscopy, Control, Incubation

Journal: Journal of Cell Science

Article Title: Macrophages show higher levels of engulfment after disruption of cis interactions between CD47 and the checkpoint receptor SIRPα

doi: 10.1242/jcs.237800

Figure Lengend Snippet: Depleting CD47 on macrophages increases engulfment activity, decreases SIRPα signaling and increases SIRPα affinity. (A) Stable knockdown of CD47 on THP-1 macrophages to 48% or 13% of wild-type (WT) levels was used to phenocopy anti-CD47 effects. Phagocytosis assays with WT or knockdown (KD) THP-1 cells used IgG-opsonized sheep RBCs (ShRBCs) or IgG-opsonized streptavidin microbeads. Microscopy fields randomly selected and 200 macrophages counted (n=3, mean±s.e.m.). Images, engulfment of ShRBCs. Arrows denote phagocytic events. Scale bar: 10 μm. THP-1 cells with 13% CD47 levels had higher levels of engulfment than WT (∼40%). *P<0.03. (B) SIRPα immunoprecipitation from lysates of THP-1 macrophages under basal conditions using anti-SIRPα (SE7C2 clone) and immunoblotted for phospho-tyrosine (pTyr; C-20 clone) (n=3, mean±s.e.m.). The normalized SIRPα pTyr signal increases linearly from zero in relation to the CD47 level. (C) (i) To quantify the effective affinity of CD47 for SIRPα in trans on WT and KD THP-1 macrophages, binding of fluorescent soluble CD47 was measured by flow cytometry and normalized to 8 μM data. All data fits y=A x/(Kd+x) (R2>0.93) for apparent dissociation constants (Kd). (ii) Kd increases linearly in relation to the CD47 level, and the non-zero intercept corresponds to the highest affinity for CD47–SIRPα as measured for human RBCs that lack SIRPα (Fig. 1B).

Article Snippet: shRNA lentiviral supernatants to CD47 were purchased from Millipore Sigma (TRC#: TRCN0000007836, TRCN0000007837) to target CD47 and resulted in 52 and 87% knockdown, respectively.

Techniques: Activity Assay, Knockdown, Microscopy, Immunoprecipitation, Binding Assay, Flow Cytometry

Journal: Journal of Cell Science

Article Title: Macrophages show higher levels of engulfment after disruption of cis interactions between CD47 and the checkpoint receptor SIRPα

doi: 10.1242/jcs.237800

Figure Lengend Snippet: Knockout of either CD47 or SIRPα on mouse B16 melanoma cells affect CD47 availability to signal ‘self’ to primary mouse macrophages. (A) A diagram of the hypothesis. Knockout of SIRPα on antibody-opsonized B16 shifts CD47 interactions from cis to trans interactions, inhibiting phagocytosis of the tumor cell. Knockout of CD47 abolishes ‘self’ signaling and increases phagocytosis. (B) Primary mouse bone marrow-derived macrophages (BMDMφs) phagocytose mouse B16 melanoma cells opsonized with anti-Tyrp1 antibody after knockout of indicated protein. Results are mean±s.d. (n=3). ***P<0.001 (one-way ANOVA and post-hoc Bonferroni test). (C) Fluorescence microscopy determinations of the phagocytic index, defined as the percentage of BMDMφs that are actively engulfing cells multiplied by the number of target cells engulfed per engulfing BMDMφ. (D) Representative images of BMDMφs engulfing opsonized B16 melanoma cells. Arrows denote phagocytic events. Scale bar: 100 μm.

Article Snippet: shRNA lentiviral supernatants to CD47 were purchased from Millipore Sigma (TRC#: TRCN0000007836, TRCN0000007837) to target CD47 and resulted in 52 and 87% knockdown, respectively.

Techniques: Knock-Out, Derivative Assay, Fluorescence, Microscopy

Journal: Journal of Cell Science

Article Title: Macrophages show higher levels of engulfment after disruption of cis interactions between CD47 and the checkpoint receptor SIRPα

doi: 10.1242/jcs.237800

Figure Lengend Snippet: In heterologous display, CD47–SIRPα trans interactions are out-competed by cis, and molecular modeling shows SIRPα bends and binds CD47 in cis. (A) Images, CHO cells expressing human CD47–GFP were labeled with anti-CD47 as detected with Alexa Fluor 647 secondary antibody. Cells co-displaying full-length human SIRPα suppress anti-CD47 binding. Scale bar: 10 μm. Diagram, anti-CD47 and soluble SIRPα bind membrane-associated CD47 in trans while competing with membrane SIRPα. (B) Quantification of anti-CD47 binding to CD47–GFP, and soluble SIRPα (labeled with red fluorophore) binding to CD47–GFP. Flow cytometry shows CD47–GFP levels vary between cells, and bound ligand signal increases proportionately. Co-displayed SIRPα reduces ligand binding in trans by >90%, consistent with cis-trans competition. Data points were generated by averaging 10,000 events from flow cytometry. (C) Top, the extracellular region of SIRPα in ribbon representation with hinge regions as yellow Cα carbons from Arg-114 to Lys-116, and Arg-220 to Phe-222. The covariance matrix of all Cα carbons is based on the first ten normal modes in a Gaussian network model; bottom, snapshots show the first three modes, highlighting rigid body domains bending and rotating around hinges. (D) Coarse-grained atomistic computation of lipid bilayer with SIRPα binding in cis to CD47 (PDB: 2JJS).

Article Snippet: shRNA lentiviral supernatants to CD47 were purchased from Millipore Sigma (TRC#: TRCN0000007836, TRCN0000007837) to target CD47 and resulted in 52 and 87% knockdown, respectively.

Techniques: Expressing, Labeling, Binding Assay, Membrane, Flow Cytometry, Ligand Binding Assay, Generated

Journal: Experimental and Therapeutic Medicine

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

doi: 10.3892/etm.2020.9054

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

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

Techniques: Flow Cytometry

Journal: Experimental and Therapeutic Medicine

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

doi: 10.3892/etm.2020.9054

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

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

Techniques: Expressing, Activation Assay, Fluorescence, FACS

Journal: Experimental and Therapeutic Medicine

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

doi: 10.3892/etm.2020.9054

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

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

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

Journal: Experimental and Therapeutic Medicine

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

doi: 10.3892/etm.2020.9054

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

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

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

Journal: Experimental and Therapeutic Medicine

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

doi: 10.3892/etm.2020.9054

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

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

Techniques: Over Expression, Expressing

Journal: Experimental and Therapeutic Medicine

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

doi: 10.3892/etm.2020.9054

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

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

Techniques: Expressing, Fluorescence

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: CD47 promotes cell growth and motility in epithelial ovarian cancer.

doi: 10.1016/j.biopha.2019.109105

Figure Lengend Snippet: Fig. 1. CD47 is highly expressed in endometriosis-associated ovarian cancer. (A, C) Immunohistochemical staining of CD47 expression in normal tissue (n = 10) and ovarian cancer (n = 36). (B) Representative image of low and high CD47 expression in ovarian cancer. (D) Quantitative immunohistochemical analysis of CD47 expression in normal tissue (n = 10), Non-EAOC (n = 26) and EAOC (n = 10). T: tumor, G: gland. Data are the mean ± SD from three independent experiments. *P < 0.05 vs. untreated control; ANOVA assay.

Article Snippet: The pcDNA3.1-CD47 plasmid was purchased from addgene (#65473), and CD47 shRNA-1 5′-GCC TTG GTT TAA TTG TGA CTT-3′ and shRNA-2 5′-GCA CAA TTA CTT GGA CTA GTT-3′ were purchased from RNAi Core.

Techniques: Immunohistochemical staining, Staining, Expressing, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: CD47 promotes cell growth and motility in epithelial ovarian cancer.

doi: 10.1016/j.biopha.2019.109105

Figure Lengend Snippet: Fig. 2. CD47 promotes cell growth of TOV-112D and TOV-21G cell lines. Cell growth was analyzed by the CCK-8 assay. Briefly, 1 μg of CD47 plasmid (A, B), 1 μg of shRNA (C, D) and 0.5 μg of antibody (E, F) were used for transfection or treatment of TOV-112D and 21G cancer cells. After 24 h, the cell growth was detected and analyzed by GraphPad prism version 6.0 software. Cell viability on the Y-axis indicates absorbance at 450 nm. Data are the mean ± SD from three independent experiments. *P < 0.05 vs. untreated control; two-tailed Student’s t-test.

Article Snippet: The pcDNA3.1-CD47 plasmid was purchased from addgene (#65473), and CD47 shRNA-1 5′-GCC TTG GTT TAA TTG TGA CTT-3′ and shRNA-2 5′-GCA CAA TTA CTT GGA CTA GTT-3′ were purchased from RNAi Core.

Techniques: CCK-8 Assay, Plasmid Preparation, shRNA, Transfection, Software, Control, Two Tailed Test

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: CD47 promotes cell growth and motility in epithelial ovarian cancer.

doi: 10.1016/j.biopha.2019.109105

Figure Lengend Snippet: Fig. 3. CD47 enhances cell migration in TOV-21G cells. Cell migration was analyzed by the wound healing assay. Briefly, 1 μg of CD47 plasmid (A, B), 1 μg of shRNA (C, D) and 0.5 μg of antibody (E, F) were used to transfect or treat the TOV-21G cancer cell line. After 24 h, cell migration was detected and the healing distance was calculated by microscopy. Data are the mean ± SD from three independent experiments. *P < 0.05 vs. untreated control; two-tailed Student’s t-test. Scale bar =200 μm.

Article Snippet: The pcDNA3.1-CD47 plasmid was purchased from addgene (#65473), and CD47 shRNA-1 5′-GCC TTG GTT TAA TTG TGA CTT-3′ and shRNA-2 5′-GCA CAA TTA CTT GGA CTA GTT-3′ were purchased from RNAi Core.

Techniques: Migration, Wound Healing Assay, Plasmid Preparation, shRNA, Microscopy, Control, Two Tailed Test

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: CD47 promotes cell growth and motility in epithelial ovarian cancer.

doi: 10.1016/j.biopha.2019.109105

Figure Lengend Snippet: Fig. 4. CD47 induces TOV-21G cell invasion. Cell invasion was analyzed by an invasion chamber. Briefly, 1 μg of CD47 plasmid (A, B), 1 μg of shRNA (C, D) and 0.5 μg of antibody (E, F) were used for transfection or treatment of TOV-21G cancer cells. After 24 h, the cell invasion was detected and calculation of invaded cells was accomplished with a microscope. Data are the mean ± SD from three independent experiments. *P < 0.05 vs. untreated control; two-tailed Student’s t-test. Scale bar =200 μm.

Article Snippet: The pcDNA3.1-CD47 plasmid was purchased from addgene (#65473), and CD47 shRNA-1 5′-GCC TTG GTT TAA TTG TGA CTT-3′ and shRNA-2 5′-GCA CAA TTA CTT GGA CTA GTT-3′ were purchased from RNAi Core.

Techniques: Plasmid Preparation, shRNA, Transfection, Microscopy, Control, Two Tailed Test

Journal: Cells

Article Title: CD47-SIRPα Signaling Induces Epithelial-Mesenchymal Transition and Cancer Stemness and Links to a Poor Prognosis in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/cells8121658

Figure Lengend Snippet: CD47 is aberrantly expressed in human oral squamous cell carcinoma and influence survival rate. ( A ) CD47 transcript expression profile across TCGA and GTEx paired normal-tumor tissue cohort. ( B ) The expression of CD47 in downloaded data for OSCC based on morphology, anatomic site, and sample type from the Genomic Data Commons-The Cancer Genome Atlas (GDC TGCA) HNSCC dataset. ( C ) Differential expression of CD47 in normal oral and cancer tissues in TCGA OSCC cohort (n = 412; p = 0.0009). ( D ) Kaplan–Meier curves showing the effect of low and high CD47 expression on the overall survival of the TGCA malignant OSCC cohort. OSCC: oral squamous cell carcinoma; GTEx: genotype-tissue expression; HNSCC: head and neck squamous cell carcinoma; GDC: genome data commons; TCGA: the cancer genome atlas.

Article Snippet: The SAS, TW2.6, HSC-3, or FaDu cells were then transfected with shRNA specifically targeting CD47 or control/scramble shRNA purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: Expressing, Quantitative Proteomics

Journal: Cells

Article Title: CD47-SIRPα Signaling Induces Epithelial-Mesenchymal Transition and Cancer Stemness and Links to a Poor Prognosis in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/cells8121658

Figure Lengend Snippet: The aberrant expression of CD47 in oral squamous cell carcinoma positively correlates with disease progression. ( A ) Representative immunohistochemistry staining of CD47 in human normal oral and OSCC tissues. ( B ) Pie chart showing the distribution of patients in our OSCC cohort (n = 71) based on histological types. ( C ) Graphical representation of the histology-specific relative expression of CD47 in tissue samples from our OSCC cohort. CD47 tissue expression is relative to that in the normal group. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The SAS, TW2.6, HSC-3, or FaDu cells were then transfected with shRNA specifically targeting CD47 or control/scramble shRNA purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: Expressing, Biomarker Discovery, Immunohistochemistry, Staining

Journal: Cells

Article Title: CD47-SIRPα Signaling Induces Epithelial-Mesenchymal Transition and Cancer Stemness and Links to a Poor Prognosis in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/cells8121658

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of clinicopathological parameters and CD47 expression for overall survival in patients with OSCC.

Article Snippet: The SAS, TW2.6, HSC-3, or FaDu cells were then transfected with shRNA specifically targeting CD47 or control/scramble shRNA purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: Expressing

Journal: Cells

Article Title: CD47-SIRPα Signaling Induces Epithelial-Mesenchymal Transition and Cancer Stemness and Links to a Poor Prognosis in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/cells8121658

Figure Lengend Snippet: CD47 modulates the cancer stem cell-like phenotype in oral squamous cell carcinoma cells. ( A ) Box and whiskers chart showing the correlative differential expression of CD47 (upper), SOX2 (middle), and CD133 (lower) mRNA from analyses of the human OSCC genome U133A array from the Toruner head-neck cohort, n = 20. ( B ) Knockdown efficiency of shCD47-1 and shCD47-2 on the protein expression of CD47 in SAS and TW2.6 cell lines shown by western blot analysis. ( C ) Effect of CD47 knockdown on the expression level of CD47, SOX2, OCT4, and CD133 proteins in SAS Sp, shCD47-1, or shCD47-2 cells shown by western blot analysis. GAPDH served as a loading control. ( D ) Immunofluorescent staining showing the effect of shCD47 on the expression of CD47, OCT4, c-Myc, and SOX2 proteins in spheres formed by TW2.6 cells. TW2.6 and SAS cells transfected with shCD47-1 or shCD47-2 exhibited decreased orosphere size (left) and number (right) in both ( E ) primary and ( F ) secondary generation orospheres. ( G ) shCD47 attenuated OCT4 and SOX2 expression and inhibited their nuclear co-localization in TW2.6- or SAS-derived orospheres, as shown by immunofluorescent (IFC) staining. All assays are representative of experiments performed four times in triplicates. WT, wild type; Sp, orosphere; blue stain = DAPI, nuclear staining. All data are representative of experiment carried out four times in triplicate and are expressed as mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The SAS, TW2.6, HSC-3, or FaDu cells were then transfected with shRNA specifically targeting CD47 or control/scramble shRNA purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: Quantitative Proteomics, Knockdown, Expressing, Western Blot, Control, Staining, Transfection, Derivative Assay

Journal: Cells

Article Title: CD47-SIRPα Signaling Induces Epithelial-Mesenchymal Transition and Cancer Stemness and Links to a Poor Prognosis in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/cells8121658

Figure Lengend Snippet: Downregulation of CD47 attenuates the EMT (epithelial-to-mesenchymal transition) and migration capacity of OSCC cells. ( A ) Representative image of scratch-wound migration assay shows the effect of shCD47 on the motility of SAS cells at 0, 3, 6, and 9-h time points (upper), and quantitative bar chart of the migrating cell fronts at indicated time points (lower). ( B ) Representative images of colony formed by WT, shCD47-1, or shCD47-2 transfected SAS cells in the culture plate using crystal violet solution and quantification of visible cells. ( C ) The inhibitory effect of shCD47 on the expression of CD47, vimentin, Slug, Snail, N-cadherin, and E-cadherin in SAS cells, as demonstrated by western blot analyses. ( D ) Photo-image, showing the fibroid/spindle shape of CD47-expressing WT cells, while shCD47 led to the loss of mesenchymal phenotype in SAS (upper) and TW2.6 (lower) cells. WT, wild type; GAPDH served as a loading control. All data are representative of experiment carried out four times in triplicate and are expressed as mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The SAS, TW2.6, HSC-3, or FaDu cells were then transfected with shRNA specifically targeting CD47 or control/scramble shRNA purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: Migration, Transfection, Expressing, Western Blot, Control

Journal: Cells

Article Title: CD47-SIRPα Signaling Induces Epithelial-Mesenchymal Transition and Cancer Stemness and Links to a Poor Prognosis in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/cells8121658

Figure Lengend Snippet: CD47 modulates the expression and subcellular localization of mesenchymal and epithelial factors in OSCC. ( A , B ) Immunofluorescent staining images, showing the expression of CD47, vimentin, and E-cadherin in SAS WT, and shCD47-1 or shCD47-2 cells. Representative dot plot of the correlative expression of ( C ) CD47 mRNA versus vimentin mRNA or ( D ) CD47 mRNA versus CDH1 mRNA in OSCC patients from the Roepman cohort, n = 220 using the R2 online genomic analysis and visualization software. ( E ) The associative network of the interaction between CD47 and molecular moieties involved in stem cell development, stem cell differentiation, stem cell maintenance, positive regulation of cell migration, and positive regulation of cell motility. Sp, orosphere; DAPI, nuclear staining.

Article Snippet: The SAS, TW2.6, HSC-3, or FaDu cells were then transfected with shRNA specifically targeting CD47 or control/scramble shRNA purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: Expressing, Staining, Software, Cell Differentiation, Migration

Journal: Cells

Article Title: CD47-SIRPα Signaling Induces Epithelial-Mesenchymal Transition and Cancer Stemness and Links to a Poor Prognosis in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/cells8121658

Figure Lengend Snippet: Suppression of CD47 expression enhances the sensitivity of OSCC-SCs to radiation therapy. ( A ) Bar chart of the inhibitory effect of exposure to 0 Gy–15 Gy radiation on the viability of SAS or TW2.6 cells. ( B ) shCD47 with or without 5 Gy–15 Gy radiation decreased the viability of SAS cells dose-dependently. ( C ) Transwell migration assay images show reduced migration in 5 Gy-exposed shCD47 SAS cells, compared to their SAS WT counterparts. ( D ) Transwell invasion assay images show reduced invasion in 5 Gy-exposed shCD47 SAS cells, compared to their SAS WT counterparts. ( E ) shCD47-transfected cells yielded smaller and fewer tumorspheres compared to their WT or shCD47 scramble counterparts. ( F ) shCD47-1 or shCD47-2 SAS cells formed fewer colonies when exposed to 5 Gy, compared to the SAS WT cells. Data represent mean ± SD from three independent experiments performed in triplicates. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The SAS, TW2.6, HSC-3, or FaDu cells were then transfected with shRNA specifically targeting CD47 or control/scramble shRNA purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: Expressing, Transwell Migration Assay, Migration, Transwell Invasion Assay, Transfection

Journal: Cells

Article Title: CD47-SIRPα Signaling Induces Epithelial-Mesenchymal Transition and Cancer Stemness and Links to a Poor Prognosis in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/cells8121658

Figure Lengend Snippet: Schema showing CD47-related molecular network and how the suppression of the ”don’t-eat-me” signal CD47 enhances radiosensitivity by downregulating cancer stem cells-associated pluripotency factors and deactivating epithelial-to-mesenchymal transition in oral squamous cell carcinoma cells.

Article Snippet: The SAS, TW2.6, HSC-3, or FaDu cells were then transfected with shRNA specifically targeting CD47 or control/scramble shRNA purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Techniques: