human breast cancer cell lines bt 474  (ATCC)


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    ATCC human breast cancer cell lines bt 474
    Human Breast Cancer Cell Lines Bt 474, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cell lines human breast cancer bt 20 atcc cat  (ATCC)


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    ATCC cell lines human breast cancer bt 20 atcc cat
    Cell Lines Human Breast Cancer Bt 20 Atcc Cat, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cell culture 97 human breast cancer cell lines bt 549  (ATCC)


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    ATCC cell culture 97 human breast cancer cell lines bt 549
    Cell Culture 97 Human Breast Cancer Cell Lines Bt 549, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human breast cancer cell line bt  (ATCC)


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    ATCC human breast cancer cell line bt
    Human Breast Cancer Cell Line Bt, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human breast cancer cell line bt 549  (ATCC)


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    ATCC human breast cancer cell line bt 549
    Human Breast Cancer Cell Line Bt 549, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human breast cancer cell lines bt 549  (ATCC)


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    ATCC human breast cancer cell lines bt 549
    Human Breast Cancer Cell Lines Bt 549, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human breast cancer cell line bt 474  (ATCC)


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    ATCC human breast cancer cell line bt 474
    Analysis of PPAB001 for simultaneous binding to CD47 and CD24 (A) Simultaneous binding of PPAB001 to recombinant CD47 and CD24 antigen by sandwich ELISA. (B) Flow cytometry of breast cancer <t>BT-474</t> cells stained with PPAB001, anti-CD24, CV1-hFc, or hIgG at 10 μg/mL. Binding kinetics of PPAB001, anti-CD24, or CV1-hFc to membrane-associated CD47 and CD24 on BT-474 cells were shown. Data are shown as means ± SDs (n = 3).
    Human Breast Cancer Cell Line Bt 474, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Dual blockade of CD47 and CD24 signaling using a novel bispecific antibody fusion protein enhances macrophage immunotherapy"

    Article Title: Dual blockade of CD47 and CD24 signaling using a novel bispecific antibody fusion protein enhances macrophage immunotherapy

    Journal: Molecular Therapy Oncolytics

    doi: 10.1016/j.omto.2023.100747

    Analysis of PPAB001 for simultaneous binding to CD47 and CD24 (A) Simultaneous binding of PPAB001 to recombinant CD47 and CD24 antigen by sandwich ELISA. (B) Flow cytometry of breast cancer BT-474 cells stained with PPAB001, anti-CD24, CV1-hFc, or hIgG at 10 μg/mL. Binding kinetics of PPAB001, anti-CD24, or CV1-hFc to membrane-associated CD47 and CD24 on BT-474 cells were shown. Data are shown as means ± SDs (n = 3).
    Figure Legend Snippet: Analysis of PPAB001 for simultaneous binding to CD47 and CD24 (A) Simultaneous binding of PPAB001 to recombinant CD47 and CD24 antigen by sandwich ELISA. (B) Flow cytometry of breast cancer BT-474 cells stained with PPAB001, anti-CD24, CV1-hFc, or hIgG at 10 μg/mL. Binding kinetics of PPAB001, anti-CD24, or CV1-hFc to membrane-associated CD47 and CD24 on BT-474 cells were shown. Data are shown as means ± SDs (n = 3).

    Techniques Used: Binding Assay, Recombinant, Sandwich ELISA, Flow Cytometry, Staining

    Treatment with PPAB001 promotes phagocytosis of human cancer cells (A) Representative phagocytosis images of macrophages engulfing BT-474, BT-474R, or SK-OV-3 cells with PPAB001, anti-CD24, CV1-hFc, or hIgG. Macrophages were stained red (CMTPX); cancer cells were labeled with green (CFSE). The white arrows indicate macrophages that engulfed cancer cells. (B) The extent of macrophage phagocytosis with PPAB001, anti-CD24, CV1-hFc, or hIgG was quantified for BT-474, BT-474R, or SK-OV-3 cells. Data are shown as means ± SDs (n = 3), and statistical significance was determined by a Student’s t test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
    Figure Legend Snippet: Treatment with PPAB001 promotes phagocytosis of human cancer cells (A) Representative phagocytosis images of macrophages engulfing BT-474, BT-474R, or SK-OV-3 cells with PPAB001, anti-CD24, CV1-hFc, or hIgG. Macrophages were stained red (CMTPX); cancer cells were labeled with green (CFSE). The white arrows indicate macrophages that engulfed cancer cells. (B) The extent of macrophage phagocytosis with PPAB001, anti-CD24, CV1-hFc, or hIgG was quantified for BT-474, BT-474R, or SK-OV-3 cells. Data are shown as means ± SDs (n = 3), and statistical significance was determined by a Student’s t test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

    Techniques Used: Staining, Labeling

    CD47/CD24 dual blockage potentiates the macrophage phagocytosis (A) Representative flow cytometry plots depicting the phagocytosis of BT-474 cells treated with PPAB001, anti-CD24 mAb, or CV1-hFc, compared with the IgG control. Phagocytosis index is shown as a bar graph. (B) Representative flow cytometry plots depicting the phagocytosis of BT-474R cells treated with PPAB001, anti-CD24 mAb, or CV1-hFc, compared with the IgG control. Phagocytosis index is shown as a bar graph. (C) Representative flow cytometry plots depicting the phagocytosis of SK-OV-3 cells treated with PPAB001, anti-CD24 mAb, or CV1-hFc, compared with the IgG control. Phagocytosis index is shown as a bar graph. Data are shown as means ± SDs (n = 3), and statistical significance was determined by a Student’s t test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
    Figure Legend Snippet: CD47/CD24 dual blockage potentiates the macrophage phagocytosis (A) Representative flow cytometry plots depicting the phagocytosis of BT-474 cells treated with PPAB001, anti-CD24 mAb, or CV1-hFc, compared with the IgG control. Phagocytosis index is shown as a bar graph. (B) Representative flow cytometry plots depicting the phagocytosis of BT-474R cells treated with PPAB001, anti-CD24 mAb, or CV1-hFc, compared with the IgG control. Phagocytosis index is shown as a bar graph. (C) Representative flow cytometry plots depicting the phagocytosis of SK-OV-3 cells treated with PPAB001, anti-CD24 mAb, or CV1-hFc, compared with the IgG control. Phagocytosis index is shown as a bar graph. Data are shown as means ± SDs (n = 3), and statistical significance was determined by a Student’s t test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

    Techniques Used: Flow Cytometry

    human breast cancer cell lines bt 549  (ATCC)


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    ATCC human breast cancer cell lines bt 549
    Human Breast Cancer Cell Lines Bt 549, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human breast cancer cell lines bt 549  (ATCC)


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    ATCC human breast cancer cell lines bt 549
    Antiproliferative effect of domperidone in TNBC cells. (A) TNBC <t>BT-549</t> and CAL-51 cells were treated with domperidone (0, 5, 10, 20, 50, and 100 µM) for 24 (black) and 48 h (gray) and cell viability was determined by WST cell viability assay. The data are shown as the mean ± SD (n=4). ** p <0.01 compared to the control group (0 µM domperidone). (B) BT-549 and CAL-51 cells treated with varying concentrations of domperidone (0, 10, 20, and 50 µM) for 24 h were analyzed by flow cytometry with annexin V/PI staining. (C) The level of cleaved forms of caspase-3, -7, -8, and -9, and PARP were monitored by immunoblot. β-Actin was used as the loading control.
    Human Breast Cancer Cell Lines Bt 549, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Domperidone Exerts Antitumor Activity in Triple-Negative Breast Cancer Cells by Modulating Reactive Oxygen Species and JAK/STAT3 Signaling"

    Article Title: Domperidone Exerts Antitumor Activity in Triple-Negative Breast Cancer Cells by Modulating Reactive Oxygen Species and JAK/STAT3 Signaling

    Journal: Biomolecules & Therapeutics

    doi: 10.4062/biomolther.2023.173

    Antiproliferative effect of domperidone in TNBC cells. (A) TNBC BT-549 and CAL-51 cells were treated with domperidone (0, 5, 10, 20, 50, and 100 µM) for 24 (black) and 48 h (gray) and cell viability was determined by WST cell viability assay. The data are shown as the mean ± SD (n=4). ** p <0.01 compared to the control group (0 µM domperidone). (B) BT-549 and CAL-51 cells treated with varying concentrations of domperidone (0, 10, 20, and 50 µM) for 24 h were analyzed by flow cytometry with annexin V/PI staining. (C) The level of cleaved forms of caspase-3, -7, -8, and -9, and PARP were monitored by immunoblot. β-Actin was used as the loading control.
    Figure Legend Snippet: Antiproliferative effect of domperidone in TNBC cells. (A) TNBC BT-549 and CAL-51 cells were treated with domperidone (0, 5, 10, 20, 50, and 100 µM) for 24 (black) and 48 h (gray) and cell viability was determined by WST cell viability assay. The data are shown as the mean ± SD (n=4). ** p <0.01 compared to the control group (0 µM domperidone). (B) BT-549 and CAL-51 cells treated with varying concentrations of domperidone (0, 10, 20, and 50 µM) for 24 h were analyzed by flow cytometry with annexin V/PI staining. (C) The level of cleaved forms of caspase-3, -7, -8, and -9, and PARP were monitored by immunoblot. β-Actin was used as the loading control.

    Techniques Used: Viability Assay, Flow Cytometry, Staining, Western Blot

    Disturbance of bcl-2 family proteins by domperidone. (A) TNBC BT-549 and CAL-51 cells were treated with domperidone (0, 10, 20, and 50 µM) for 24 h and the levels of Bcl-2 family proteins Bcl-2, Bcl-xL, Bax, and Bak were monitored by immunoblot. β-Actin was used as the loading control. (B) The mitochondrial membrane potential was measured by flow cytometry with DiOC6 staining. The bar graphs represent the mean ± SD (n=3). * p <0.05 compared to the control group.
    Figure Legend Snippet: Disturbance of bcl-2 family proteins by domperidone. (A) TNBC BT-549 and CAL-51 cells were treated with domperidone (0, 10, 20, and 50 µM) for 24 h and the levels of Bcl-2 family proteins Bcl-2, Bcl-xL, Bax, and Bak were monitored by immunoblot. β-Actin was used as the loading control. (B) The mitochondrial membrane potential was measured by flow cytometry with DiOC6 staining. The bar graphs represent the mean ± SD (n=3). * p <0.05 compared to the control group.

    Techniques Used: Western Blot, Membrane, Flow Cytometry, Staining

    Induction of mitochondrial ROS by domperidone. (A) TNBC BT-549 and CAL-51 cells were treated with domperidone (0, 10, 20, and 50 µM) for 24 h and the levels of mitochondrial superoxide were analyzed by flow cytometry with MitoSOX TM Red staining. The bar graphs represent the mean ± SD (n=3). * p <0.05 and ** p <0.01 compared to the control group. (B) TNBC cells were pretreated with 100 nM of Mito-TEMPO for 3 h before domperidone treatment (100 µM for 24 h). The cell viability was measured by WST assay. ** p <0.01 compared to the domperidone treated group.
    Figure Legend Snippet: Induction of mitochondrial ROS by domperidone. (A) TNBC BT-549 and CAL-51 cells were treated with domperidone (0, 10, 20, and 50 µM) for 24 h and the levels of mitochondrial superoxide were analyzed by flow cytometry with MitoSOX TM Red staining. The bar graphs represent the mean ± SD (n=3). * p <0.05 and ** p <0.01 compared to the control group. (B) TNBC cells were pretreated with 100 nM of Mito-TEMPO for 3 h before domperidone treatment (100 µM for 24 h). The cell viability was measured by WST assay. ** p <0.01 compared to the domperidone treated group.

    Techniques Used: Flow Cytometry, Staining, WST Assay

    Interference of cell cycle progression by domperidone. TNBC BT-549 and CAL-51 cells were treated with domperidone (0, 10, 20, and 50 µM) for 24 h. (A) Cell cycle analysis by flow cytometry with PI staining. (B) The immunoblot analysis of cell cycle related proteins cyclins A, B, D1, D2, D3, and E, and CDK1, 2, and 4. β-Actin was used as the loading control. (C) The immunoblot analysis of CDK inhibitors p16, p21, and p27, and p53.
    Figure Legend Snippet: Interference of cell cycle progression by domperidone. TNBC BT-549 and CAL-51 cells were treated with domperidone (0, 10, 20, and 50 µM) for 24 h. (A) Cell cycle analysis by flow cytometry with PI staining. (B) The immunoblot analysis of cell cycle related proteins cyclins A, B, D1, D2, D3, and E, and CDK1, 2, and 4. β-Actin was used as the loading control. (C) The immunoblot analysis of CDK inhibitors p16, p21, and p27, and p53.

    Techniques Used: Cell Cycle Assay, Flow Cytometry, Staining, Western Blot

    Regulation of JAK2/STAT3 signaling by domperidone. TNBC BT-549 and CAL-51 cells were treated with domperidone (0, 10, 20, and 50 µM) for 24 h. (A) The immunoblot analysis of proteins involved in Jak/STAT signaling pathways. (B) The immunoblot analysis of proteins involved with MAP kinase signaling pathway. β-Actin was used as the loading control.
    Figure Legend Snippet: Regulation of JAK2/STAT3 signaling by domperidone. TNBC BT-549 and CAL-51 cells were treated with domperidone (0, 10, 20, and 50 µM) for 24 h. (A) The immunoblot analysis of proteins involved in Jak/STAT signaling pathways. (B) The immunoblot analysis of proteins involved with MAP kinase signaling pathway. β-Actin was used as the loading control.

    Techniques Used: Western Blot

    Downregulation of the dopamine D2-like receptor by domperidone. (A) TNBC BT-549 and CAL-51 cells were treated with domperidone (0, 10, 20, and 50 µM) for 24 h. The immunoblot analysis of dopamine receptors DRD1 through DRD5. β-Actin was used as the loading control. (B) The fold of induction in the mRNA level analyzed by reverse transcription polymerase chain reaction. Black bars: control (0 µM domperidone). Grey bars: domperidone treatment (50 µM) for 24 h. ** p <0.01 compared to the non-treated control. ND, not detected.
    Figure Legend Snippet: Downregulation of the dopamine D2-like receptor by domperidone. (A) TNBC BT-549 and CAL-51 cells were treated with domperidone (0, 10, 20, and 50 µM) for 24 h. The immunoblot analysis of dopamine receptors DRD1 through DRD5. β-Actin was used as the loading control. (B) The fold of induction in the mRNA level analyzed by reverse transcription polymerase chain reaction. Black bars: control (0 µM domperidone). Grey bars: domperidone treatment (50 µM) for 24 h. ** p <0.01 compared to the non-treated control. ND, not detected.

    Techniques Used: Western Blot, Reverse Transcription Polymerase Chain Reaction

    human breast cancer cell lines bt 549  (ATCC)


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    ATCC human breast cancer cell lines bt 549
    SNHG15 promotes the proliferation and invasion of breast cancer cells. ( A ) IMP1 antibody was used to perform RIP assays in <t>BT-549</t> cells. Western blots (left panel) and RT-qPCR (right panel) show that IMP1 was truly associated with SNHG15 and five other selected lncRNAs. *** p < 0.001, ** p < 0.01 and * p < 0.05. ( B ) RT-PCR and gel electrophoresis show that the 983 bp (V4) SHNG15 is a major transcript in breast cancer cells. ( C ) RT-PCR and gel electrophoresis indicate the relative levels of endogenous SNHG15 in four breast cancer cell lines. GADP mRNA was used as an internal control. ( D ) Cell proliferation assays were performed in T47D cells, which showed that knocking down SNHG15 expression decreased cell growth potential. ** p < 0.01. ( E ) Transwell assays indicated that over-expression of SNHG15 in BT-549 cells increased cell invasive ability. *** p < 0.001.
    Human Breast Cancer Cell Lines Bt 549, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "SNHG15-Mediated Localization of Nucleolin at the Cell Protrusions Regulates CDH2 mRNA Expression and Cell Invasion"

    Article Title: SNHG15-Mediated Localization of Nucleolin at the Cell Protrusions Regulates CDH2 mRNA Expression and Cell Invasion

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms242115600

    SNHG15 promotes the proliferation and invasion of breast cancer cells. ( A ) IMP1 antibody was used to perform RIP assays in BT-549 cells. Western blots (left panel) and RT-qPCR (right panel) show that IMP1 was truly associated with SNHG15 and five other selected lncRNAs. *** p < 0.001, ** p < 0.01 and * p < 0.05. ( B ) RT-PCR and gel electrophoresis show that the 983 bp (V4) SHNG15 is a major transcript in breast cancer cells. ( C ) RT-PCR and gel electrophoresis indicate the relative levels of endogenous SNHG15 in four breast cancer cell lines. GADP mRNA was used as an internal control. ( D ) Cell proliferation assays were performed in T47D cells, which showed that knocking down SNHG15 expression decreased cell growth potential. ** p < 0.01. ( E ) Transwell assays indicated that over-expression of SNHG15 in BT-549 cells increased cell invasive ability. *** p < 0.001.
    Figure Legend Snippet: SNHG15 promotes the proliferation and invasion of breast cancer cells. ( A ) IMP1 antibody was used to perform RIP assays in BT-549 cells. Western blots (left panel) and RT-qPCR (right panel) show that IMP1 was truly associated with SNHG15 and five other selected lncRNAs. *** p < 0.001, ** p < 0.01 and * p < 0.05. ( B ) RT-PCR and gel electrophoresis show that the 983 bp (V4) SHNG15 is a major transcript in breast cancer cells. ( C ) RT-PCR and gel electrophoresis indicate the relative levels of endogenous SNHG15 in four breast cancer cell lines. GADP mRNA was used as an internal control. ( D ) Cell proliferation assays were performed in T47D cells, which showed that knocking down SNHG15 expression decreased cell growth potential. ** p < 0.01. ( E ) Transwell assays indicated that over-expression of SNHG15 in BT-549 cells increased cell invasive ability. *** p < 0.001.

    Techniques Used: Western Blot, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Expressing, Over Expression

    IMP1 regulates the localization of SNHG15 at the cell protrusions. ( A ) FISH was performed to detect the subcellular localization of SNHG15 in WT BT-549 cells (upper panel) and SNHG15-expressing BT-549 cells (lower panel). The arrowheads indicate localized SNHG15 at the cell protrusions. Scale bar: 10 µm. ( B ) FISH indicated that, in comparison with WT MDA-MB-231 cells (upper panel), IMP1 expression greatly increased the localization of SHNG15 at cell protrusions (lower panel). Scale bar: 10 µm. ( C ) A bar graph indicates the percentage of protrusion-localized SNHG15 in tested cells. Localization was increased to 70% from 40% when ectopic IMP1 was expressed. About 80–100 cells were counted in each group. ** p < 0.01. WT: cell origin. IMP1: cells expressing ectopic IMP1. Control: cells transfected with an empty plasmid. ( D ) RT-qPCR showed that IMP1 expression does not affect cellular levels of SNHG15.
    Figure Legend Snippet: IMP1 regulates the localization of SNHG15 at the cell protrusions. ( A ) FISH was performed to detect the subcellular localization of SNHG15 in WT BT-549 cells (upper panel) and SNHG15-expressing BT-549 cells (lower panel). The arrowheads indicate localized SNHG15 at the cell protrusions. Scale bar: 10 µm. ( B ) FISH indicated that, in comparison with WT MDA-MB-231 cells (upper panel), IMP1 expression greatly increased the localization of SHNG15 at cell protrusions (lower panel). Scale bar: 10 µm. ( C ) A bar graph indicates the percentage of protrusion-localized SNHG15 in tested cells. Localization was increased to 70% from 40% when ectopic IMP1 was expressed. About 80–100 cells were counted in each group. ** p < 0.01. WT: cell origin. IMP1: cells expressing ectopic IMP1. Control: cells transfected with an empty plasmid. ( D ) RT-qPCR showed that IMP1 expression does not affect cellular levels of SNHG15.

    Techniques Used: Expressing, Comparison, Transfection, Plasmid Preparation, Quantitative RT-PCR

    SNHG15 forms a complex with nucleolin and carries nucleolin to the cell protrusions. ( A ) RIP experiments using antibodies against human nucleolin were performed. Western blots and RT-PCR indicated that SNHG15 forms a complex with nucleolin. ( B ) A bar graph of IF assays indicates that accumulated nucleolin at the protrusions was greatly increased in SNHG15-expressing cells. *** p < 0.001. ( C ) IF assays were performed in BT-549 WT cells and cells expressing SNHG15 using nucleolin antibody. The accumulation of nucleolin at the cell protrusions was strongly increased in SNHG15-expressing cells. The arrowheads indicate protrusion localized nucleolin. ( D ) FISH and IF double staining assays showed that SNHG15 and nucleolin were co-localized (arrowheads indicated) at the cell protrusions of BT-549 cells. Scale bar: 10 µm.
    Figure Legend Snippet: SNHG15 forms a complex with nucleolin and carries nucleolin to the cell protrusions. ( A ) RIP experiments using antibodies against human nucleolin were performed. Western blots and RT-PCR indicated that SNHG15 forms a complex with nucleolin. ( B ) A bar graph of IF assays indicates that accumulated nucleolin at the protrusions was greatly increased in SNHG15-expressing cells. *** p < 0.001. ( C ) IF assays were performed in BT-549 WT cells and cells expressing SNHG15 using nucleolin antibody. The accumulation of nucleolin at the cell protrusions was strongly increased in SNHG15-expressing cells. The arrowheads indicate protrusion localized nucleolin. ( D ) FISH and IF double staining assays showed that SNHG15 and nucleolin were co-localized (arrowheads indicated) at the cell protrusions of BT-549 cells. Scale bar: 10 µm.

    Techniques Used: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Double Staining

    Expression of CDH2 mRNA was altered in responding to SNHG15 expression. ( A ) Six individual transcripts were selected to confirm their expression in response to SNHG15 expression in BT-549 cell lines. qRT-PCR showed that all selected mRNAs, including CDH2 mRNA, displayed a similar differential expression pattern, as indicated in BT-549 cells. ** p < 0.01, * p < 0.1. ( B ) FISH showed that substantial CDH2 mRNA was localized at the cell protrusion in SNHG15-expressing cells. The arrowheads indicate CDH2 mRNAs. Scale bar: 10 µm. ( C , D ) IF indicated that about 70% of cells showed CDH2 protein that was predominantly accumulated at the cell protrusions when SNHG15 was expressed. The arrowheads indicate detected CDH2 protein. Scale bar: 10 µm. ( E ) SNHG15 increased CDH2 but not CDH1 expression. Immunoblot analysis of proteins isolated from BT-549 cells with or without SNHG15 expression was performed. Numbers below the bands indicate relative levels of CDH2 and CDH1 proteins, which were normalized to GADH protein.
    Figure Legend Snippet: Expression of CDH2 mRNA was altered in responding to SNHG15 expression. ( A ) Six individual transcripts were selected to confirm their expression in response to SNHG15 expression in BT-549 cell lines. qRT-PCR showed that all selected mRNAs, including CDH2 mRNA, displayed a similar differential expression pattern, as indicated in BT-549 cells. ** p < 0.01, * p < 0.1. ( B ) FISH showed that substantial CDH2 mRNA was localized at the cell protrusion in SNHG15-expressing cells. The arrowheads indicate CDH2 mRNAs. Scale bar: 10 µm. ( C , D ) IF indicated that about 70% of cells showed CDH2 protein that was predominantly accumulated at the cell protrusions when SNHG15 was expressed. The arrowheads indicate detected CDH2 protein. Scale bar: 10 µm. ( E ) SNHG15 increased CDH2 but not CDH1 expression. Immunoblot analysis of proteins isolated from BT-549 cells with or without SNHG15 expression was performed. Numbers below the bands indicate relative levels of CDH2 and CDH1 proteins, which were normalized to GADH protein.

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Isolation

    Accumulation of nucleolin at the cell protrusion enhances local translation of CDH2 mRNA and increases cell invasive potential. ( A ) Nucleolin antibody was used for immunoprecipitation of nucleolin and its associated RNA targets in breast cancer cells. Normal IgG and GAPDH were used as negative controls. *** p < 0.001. ( B ) CDH2 mRNA was preferentially bound to nucleolin. ( C , D ) IF and FISH double staining indicated that, in SNHG15-expressing cells, about 70% of cells showed strong co-localization of CDH2 mRNA and nucleolin at the cell protrusions (Arrowheads indicated). Scale bar: 10 µm. ( E ) Upper: the 3′UTR of CDH2 mRNA was cloned into the downstream region of the renilla luciferase gene of the PsiCHECK 2 dual luciferase reporter; lower: the reporter was transfected into SNHG15-expressing BT-549 cells for 48 h. Luciferase activity increased in cells expressing SNHG15. ( F ) The reporter was transfected into SNHG15-silenced MDA-MB-231 cells for 48 h. Luciferase activity was reduced when SNHG15 was knocked down by siRNA. ** p < 0.01. ( G ) Transwell assays were performed in SNHG15-expressing BT-549 cells in which SNHG15 or CDH2 mRNA was knocked down by corresponding siRNAs. ** p < 0.01. ( H ) Transwell assays were performed in MDA-MB-231 cells in which endogenous SNHG15 was knocked down by siRNA. ** p < 0.01.
    Figure Legend Snippet: Accumulation of nucleolin at the cell protrusion enhances local translation of CDH2 mRNA and increases cell invasive potential. ( A ) Nucleolin antibody was used for immunoprecipitation of nucleolin and its associated RNA targets in breast cancer cells. Normal IgG and GAPDH were used as negative controls. *** p < 0.001. ( B ) CDH2 mRNA was preferentially bound to nucleolin. ( C , D ) IF and FISH double staining indicated that, in SNHG15-expressing cells, about 70% of cells showed strong co-localization of CDH2 mRNA and nucleolin at the cell protrusions (Arrowheads indicated). Scale bar: 10 µm. ( E ) Upper: the 3′UTR of CDH2 mRNA was cloned into the downstream region of the renilla luciferase gene of the PsiCHECK 2 dual luciferase reporter; lower: the reporter was transfected into SNHG15-expressing BT-549 cells for 48 h. Luciferase activity increased in cells expressing SNHG15. ( F ) The reporter was transfected into SNHG15-silenced MDA-MB-231 cells for 48 h. Luciferase activity was reduced when SNHG15 was knocked down by siRNA. ** p < 0.01. ( G ) Transwell assays were performed in SNHG15-expressing BT-549 cells in which SNHG15 or CDH2 mRNA was knocked down by corresponding siRNAs. ** p < 0.01. ( H ) Transwell assays were performed in MDA-MB-231 cells in which endogenous SNHG15 was knocked down by siRNA. ** p < 0.01.

    Techniques Used: Immunoprecipitation, Double Staining, Expressing, Clone Assay, Luciferase, Transfection, Activity Assay

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    ATCC human breast cancer cell lines bt 474
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    ATCC human breast cancer cell line bt 474
    Analysis of PPAB001 for simultaneous binding to CD47 and CD24 (A) Simultaneous binding of PPAB001 to recombinant CD47 and CD24 antigen by sandwich ELISA. (B) Flow cytometry of breast cancer <t>BT-474</t> cells stained with PPAB001, anti-CD24, CV1-hFc, or hIgG at 10 μg/mL. Binding kinetics of PPAB001, anti-CD24, or CV1-hFc to membrane-associated CD47 and CD24 on BT-474 cells were shown. Data are shown as means ± SDs (n = 3).
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    Analysis of PPAB001 for simultaneous binding to CD47 and CD24 (A) Simultaneous binding of PPAB001 to recombinant CD47 and CD24 antigen by sandwich ELISA. (B) Flow cytometry of breast cancer BT-474 cells stained with PPAB001, anti-CD24, CV1-hFc, or hIgG at 10 μg/mL. Binding kinetics of PPAB001, anti-CD24, or CV1-hFc to membrane-associated CD47 and CD24 on BT-474 cells were shown. Data are shown as means ± SDs (n = 3).

    Journal: Molecular Therapy Oncolytics

    Article Title: Dual blockade of CD47 and CD24 signaling using a novel bispecific antibody fusion protein enhances macrophage immunotherapy

    doi: 10.1016/j.omto.2023.100747

    Figure Lengend Snippet: Analysis of PPAB001 for simultaneous binding to CD47 and CD24 (A) Simultaneous binding of PPAB001 to recombinant CD47 and CD24 antigen by sandwich ELISA. (B) Flow cytometry of breast cancer BT-474 cells stained with PPAB001, anti-CD24, CV1-hFc, or hIgG at 10 μg/mL. Binding kinetics of PPAB001, anti-CD24, or CV1-hFc to membrane-associated CD47 and CD24 on BT-474 cells were shown. Data are shown as means ± SDs (n = 3).

    Article Snippet: The human breast cancer cell line BT-474 and the ovarian cancer cell line SK-OV-3 were obtained from the American Type Culture Collection (ATCC).

    Techniques: Binding Assay, Recombinant, Sandwich ELISA, Flow Cytometry, Staining

    Treatment with PPAB001 promotes phagocytosis of human cancer cells (A) Representative phagocytosis images of macrophages engulfing BT-474, BT-474R, or SK-OV-3 cells with PPAB001, anti-CD24, CV1-hFc, or hIgG. Macrophages were stained red (CMTPX); cancer cells were labeled with green (CFSE). The white arrows indicate macrophages that engulfed cancer cells. (B) The extent of macrophage phagocytosis with PPAB001, anti-CD24, CV1-hFc, or hIgG was quantified for BT-474, BT-474R, or SK-OV-3 cells. Data are shown as means ± SDs (n = 3), and statistical significance was determined by a Student’s t test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

    Journal: Molecular Therapy Oncolytics

    Article Title: Dual blockade of CD47 and CD24 signaling using a novel bispecific antibody fusion protein enhances macrophage immunotherapy

    doi: 10.1016/j.omto.2023.100747

    Figure Lengend Snippet: Treatment with PPAB001 promotes phagocytosis of human cancer cells (A) Representative phagocytosis images of macrophages engulfing BT-474, BT-474R, or SK-OV-3 cells with PPAB001, anti-CD24, CV1-hFc, or hIgG. Macrophages were stained red (CMTPX); cancer cells were labeled with green (CFSE). The white arrows indicate macrophages that engulfed cancer cells. (B) The extent of macrophage phagocytosis with PPAB001, anti-CD24, CV1-hFc, or hIgG was quantified for BT-474, BT-474R, or SK-OV-3 cells. Data are shown as means ± SDs (n = 3), and statistical significance was determined by a Student’s t test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

    Article Snippet: The human breast cancer cell line BT-474 and the ovarian cancer cell line SK-OV-3 were obtained from the American Type Culture Collection (ATCC).

    Techniques: Staining, Labeling

    CD47/CD24 dual blockage potentiates the macrophage phagocytosis (A) Representative flow cytometry plots depicting the phagocytosis of BT-474 cells treated with PPAB001, anti-CD24 mAb, or CV1-hFc, compared with the IgG control. Phagocytosis index is shown as a bar graph. (B) Representative flow cytometry plots depicting the phagocytosis of BT-474R cells treated with PPAB001, anti-CD24 mAb, or CV1-hFc, compared with the IgG control. Phagocytosis index is shown as a bar graph. (C) Representative flow cytometry plots depicting the phagocytosis of SK-OV-3 cells treated with PPAB001, anti-CD24 mAb, or CV1-hFc, compared with the IgG control. Phagocytosis index is shown as a bar graph. Data are shown as means ± SDs (n = 3), and statistical significance was determined by a Student’s t test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

    Journal: Molecular Therapy Oncolytics

    Article Title: Dual blockade of CD47 and CD24 signaling using a novel bispecific antibody fusion protein enhances macrophage immunotherapy

    doi: 10.1016/j.omto.2023.100747

    Figure Lengend Snippet: CD47/CD24 dual blockage potentiates the macrophage phagocytosis (A) Representative flow cytometry plots depicting the phagocytosis of BT-474 cells treated with PPAB001, anti-CD24 mAb, or CV1-hFc, compared with the IgG control. Phagocytosis index is shown as a bar graph. (B) Representative flow cytometry plots depicting the phagocytosis of BT-474R cells treated with PPAB001, anti-CD24 mAb, or CV1-hFc, compared with the IgG control. Phagocytosis index is shown as a bar graph. (C) Representative flow cytometry plots depicting the phagocytosis of SK-OV-3 cells treated with PPAB001, anti-CD24 mAb, or CV1-hFc, compared with the IgG control. Phagocytosis index is shown as a bar graph. Data are shown as means ± SDs (n = 3), and statistical significance was determined by a Student’s t test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

    Article Snippet: The human breast cancer cell line BT-474 and the ovarian cancer cell line SK-OV-3 were obtained from the American Type Culture Collection (ATCC).

    Techniques: Flow Cytometry