cd47 promoter wt plasmid  (Santa Cruz Biotechnology)

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