knockdown bmi1 gene  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc knockdown bmi1 gene
    A Representative images of <t>BMI1</t> staining (green bodies). Nuclei are counterstained with DAPi (blue staining). BMI1 protein can accumulate in CAP bodies (left panel) or has uniform punctate distribution (PcG bodies, right panel). Scale bar: 5 μm. B Stacked bar plot representing the proportion of CAP and PcG bodies in ALDH br and ALDH neg SUM159 cells sorted according to their cell-cycle phase (G0/G1, S, and G2/M). C , D Representative images (left panels) of BMI1 costaining (green foci) with γH2AX (red foci) for cells harboring CAP bodies ( C ) or PcG bodies ( D ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and γH2AX fluorescent signals. E Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/γH2AX colocalization. F , G Representative images (left panels) of BMI1 costaining (green foci) with RAD51 (red foci) for cells harboring CAP bodies ( F ) or PcG bodies ( G ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and RAD51 fluorescent signals. H Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/RAD51 colocalization. I Representative images of BMI1 staining (green bodies) in ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated (CTRL). Nuclei are counterstained with DAPi (blue staining). J Stacked bar plot representing the proportion of CAP and PcG bodies in replicative ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated. K Bar plots representing the proportion of γH2AX-positive cells for each cell subpopulation (replicative ALDH br and ALDH neg SUM159 cells) following 5-aza treatment or in untreated conditions (CTRL). Statistical test used is Student’s t -test. Data represent mean ± SD.
    Knockdown Bmi1 Gene, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "BMI1 nuclear location is critical for RAD51-dependent response to replication stress and drives chemoresistance in breast cancer stem cells"

    Article Title: BMI1 nuclear location is critical for RAD51-dependent response to replication stress and drives chemoresistance in breast cancer stem cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-04538-w

    A Representative images of BMI1 staining (green bodies). Nuclei are counterstained with DAPi (blue staining). BMI1 protein can accumulate in CAP bodies (left panel) or has uniform punctate distribution (PcG bodies, right panel). Scale bar: 5 μm. B Stacked bar plot representing the proportion of CAP and PcG bodies in ALDH br and ALDH neg SUM159 cells sorted according to their cell-cycle phase (G0/G1, S, and G2/M). C , D Representative images (left panels) of BMI1 costaining (green foci) with γH2AX (red foci) for cells harboring CAP bodies ( C ) or PcG bodies ( D ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and γH2AX fluorescent signals. E Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/γH2AX colocalization. F , G Representative images (left panels) of BMI1 costaining (green foci) with RAD51 (red foci) for cells harboring CAP bodies ( F ) or PcG bodies ( G ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and RAD51 fluorescent signals. H Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/RAD51 colocalization. I Representative images of BMI1 staining (green bodies) in ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated (CTRL). Nuclei are counterstained with DAPi (blue staining). J Stacked bar plot representing the proportion of CAP and PcG bodies in replicative ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated. K Bar plots representing the proportion of γH2AX-positive cells for each cell subpopulation (replicative ALDH br and ALDH neg SUM159 cells) following 5-aza treatment or in untreated conditions (CTRL). Statistical test used is Student’s t -test. Data represent mean ± SD.
    Figure Legend Snippet: A Representative images of BMI1 staining (green bodies). Nuclei are counterstained with DAPi (blue staining). BMI1 protein can accumulate in CAP bodies (left panel) or has uniform punctate distribution (PcG bodies, right panel). Scale bar: 5 μm. B Stacked bar plot representing the proportion of CAP and PcG bodies in ALDH br and ALDH neg SUM159 cells sorted according to their cell-cycle phase (G0/G1, S, and G2/M). C , D Representative images (left panels) of BMI1 costaining (green foci) with γH2AX (red foci) for cells harboring CAP bodies ( C ) or PcG bodies ( D ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and γH2AX fluorescent signals. E Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/γH2AX colocalization. F , G Representative images (left panels) of BMI1 costaining (green foci) with RAD51 (red foci) for cells harboring CAP bodies ( F ) or PcG bodies ( G ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and RAD51 fluorescent signals. H Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/RAD51 colocalization. I Representative images of BMI1 staining (green bodies) in ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated (CTRL). Nuclei are counterstained with DAPi (blue staining). J Stacked bar plot representing the proportion of CAP and PcG bodies in replicative ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated. K Bar plots representing the proportion of γH2AX-positive cells for each cell subpopulation (replicative ALDH br and ALDH neg SUM159 cells) following 5-aza treatment or in untreated conditions (CTRL). Statistical test used is Student’s t -test. Data represent mean ± SD.

    Techniques Used: Staining

    knockdown bmi1 gene  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc knockdown bmi1 gene
    A Representative images of <t>BMI1</t> staining (green bodies). Nuclei are counterstained with DAPi (blue staining). BMI1 protein can accumulate in CAP bodies (left panel) or has uniform punctate distribution (PcG bodies, right panel). Scale bar: 5 μm. B Stacked bar plot representing the proportion of CAP and PcG bodies in ALDH br and ALDH neg SUM159 cells sorted according to their cell-cycle phase (G0/G1, S, and G2/M). C , D Representative images (left panels) of BMI1 costaining (green foci) with γH2AX (red foci) for cells harboring CAP bodies ( C ) or PcG bodies ( D ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and γH2AX fluorescent signals. E Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/γH2AX colocalization. F , G Representative images (left panels) of BMI1 costaining (green foci) with RAD51 (red foci) for cells harboring CAP bodies ( F ) or PcG bodies ( G ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and RAD51 fluorescent signals. H Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/RAD51 colocalization. I Representative images of BMI1 staining (green bodies) in ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated (CTRL). Nuclei are counterstained with DAPi (blue staining). J Stacked bar plot representing the proportion of CAP and PcG bodies in replicative ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated. K Bar plots representing the proportion of γH2AX-positive cells for each cell subpopulation (replicative ALDH br and ALDH neg SUM159 cells) following 5-aza treatment or in untreated conditions (CTRL). Statistical test used is Student’s t -test. Data represent mean ± SD.
    Knockdown Bmi1 Gene, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/knockdown bmi1 gene/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    knockdown bmi1 gene - by Bioz Stars, 2023-02
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    Images

    1) Product Images from "BMI1 nuclear location is critical for RAD51-dependent response to replication stress and drives chemoresistance in breast cancer stem cells"

    Article Title: BMI1 nuclear location is critical for RAD51-dependent response to replication stress and drives chemoresistance in breast cancer stem cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-04538-w

    A Representative images of BMI1 staining (green bodies). Nuclei are counterstained with DAPi (blue staining). BMI1 protein can accumulate in CAP bodies (left panel) or has uniform punctate distribution (PcG bodies, right panel). Scale bar: 5 μm. B Stacked bar plot representing the proportion of CAP and PcG bodies in ALDH br and ALDH neg SUM159 cells sorted according to their cell-cycle phase (G0/G1, S, and G2/M). C , D Representative images (left panels) of BMI1 costaining (green foci) with γH2AX (red foci) for cells harboring CAP bodies ( C ) or PcG bodies ( D ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and γH2AX fluorescent signals. E Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/γH2AX colocalization. F , G Representative images (left panels) of BMI1 costaining (green foci) with RAD51 (red foci) for cells harboring CAP bodies ( F ) or PcG bodies ( G ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and RAD51 fluorescent signals. H Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/RAD51 colocalization. I Representative images of BMI1 staining (green bodies) in ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated (CTRL). Nuclei are counterstained with DAPi (blue staining). J Stacked bar plot representing the proportion of CAP and PcG bodies in replicative ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated. K Bar plots representing the proportion of γH2AX-positive cells for each cell subpopulation (replicative ALDH br and ALDH neg SUM159 cells) following 5-aza treatment or in untreated conditions (CTRL). Statistical test used is Student’s t -test. Data represent mean ± SD.
    Figure Legend Snippet: A Representative images of BMI1 staining (green bodies). Nuclei are counterstained with DAPi (blue staining). BMI1 protein can accumulate in CAP bodies (left panel) or has uniform punctate distribution (PcG bodies, right panel). Scale bar: 5 μm. B Stacked bar plot representing the proportion of CAP and PcG bodies in ALDH br and ALDH neg SUM159 cells sorted according to their cell-cycle phase (G0/G1, S, and G2/M). C , D Representative images (left panels) of BMI1 costaining (green foci) with γH2AX (red foci) for cells harboring CAP bodies ( C ) or PcG bodies ( D ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and γH2AX fluorescent signals. E Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/γH2AX colocalization. F , G Representative images (left panels) of BMI1 costaining (green foci) with RAD51 (red foci) for cells harboring CAP bodies ( F ) or PcG bodies ( G ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and RAD51 fluorescent signals. H Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/RAD51 colocalization. I Representative images of BMI1 staining (green bodies) in ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated (CTRL). Nuclei are counterstained with DAPi (blue staining). J Stacked bar plot representing the proportion of CAP and PcG bodies in replicative ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated. K Bar plots representing the proportion of γH2AX-positive cells for each cell subpopulation (replicative ALDH br and ALDH neg SUM159 cells) following 5-aza treatment or in untreated conditions (CTRL). Statistical test used is Student’s t -test. Data represent mean ± SD.

    Techniques Used: Staining

    bmi1 sirna  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc bmi1 sirna
    A–C. BT-549 cells were stably transduced to express a tetracycline-inducible control shRNA (tet-CshRNA) (A) or a MUC1 shRNA (tet-MUC1shRNA) (B). Cells treated with 200 ng/ml DOX for 4 d were analyzed for MUC1 and <t>BMI1</t> mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1). Cell lysates treated with 200 ng/ml DOX for 7 d were immunoblotted with the indicated antibodies (C). D. MDA-MB-231/tet-MUC1shRNA cells treated with 200 ng/ml DOX for 4 d were analyzed for MUC1 and BMI1 mRNA levels by qRT-PCR (left). Cell lysates treated with 200 ng/ml DOX for 7 d were immunoblotted with the indicated antibodies (right). E. BT-20 cells stably expressing a control or MUC1-C vector were analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for vector cells (assigned a value of 1) (left). Lysates were immunoblotted with the indicated antibodies (right).
    Bmi1 Sirna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bmi1 sirna/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bmi1 sirna - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "MUC1-C ACTIVATES BMI1 IN HUMAN CANCER CELLS"

    Article Title: MUC1-C ACTIVATES BMI1 IN HUMAN CANCER CELLS

    Journal: Oncogene

    doi: 10.1038/onc.2016.439

    A–C. BT-549 cells were stably transduced to express a tetracycline-inducible control shRNA (tet-CshRNA) (A) or a MUC1 shRNA (tet-MUC1shRNA) (B). Cells treated with 200 ng/ml DOX for 4 d were analyzed for MUC1 and BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1). Cell lysates treated with 200 ng/ml DOX for 7 d were immunoblotted with the indicated antibodies (C). D. MDA-MB-231/tet-MUC1shRNA cells treated with 200 ng/ml DOX for 4 d were analyzed for MUC1 and BMI1 mRNA levels by qRT-PCR (left). Cell lysates treated with 200 ng/ml DOX for 7 d were immunoblotted with the indicated antibodies (right). E. BT-20 cells stably expressing a control or MUC1-C vector were analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for vector cells (assigned a value of 1) (left). Lysates were immunoblotted with the indicated antibodies (right).
    Figure Legend Snippet: A–C. BT-549 cells were stably transduced to express a tetracycline-inducible control shRNA (tet-CshRNA) (A) or a MUC1 shRNA (tet-MUC1shRNA) (B). Cells treated with 200 ng/ml DOX for 4 d were analyzed for MUC1 and BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1). Cell lysates treated with 200 ng/ml DOX for 7 d were immunoblotted with the indicated antibodies (C). D. MDA-MB-231/tet-MUC1shRNA cells treated with 200 ng/ml DOX for 4 d were analyzed for MUC1 and BMI1 mRNA levels by qRT-PCR (left). Cell lysates treated with 200 ng/ml DOX for 7 d were immunoblotted with the indicated antibodies (right). E. BT-20 cells stably expressing a control or MUC1-C vector were analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for vector cells (assigned a value of 1) (left). Lysates were immunoblotted with the indicated antibodies (right).

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

    A. Schema of the MUC1-C subunit with the 58 aa extracellular domain (ED), the 28 aa transmembrane domain (TM), and the sequence of the 72 aa cytoplasmic domain (CD). The MUC1-C cytoplasmic domain contains a CQC motif that is necessary and sufficient for MUC1-C homodimerization and oncogenic function. GO-203 is a cell-penetrating peptide that binds the CQC motif and blocks MUC1-C homodimerization. Highlighted are MUC1-C-induced pathways that confer the activation of ZEB1 and MYC. B. BT-549 cells were transfected with a control or MUC1-C(AQA) vector in which the CQC motif had been mutated to AQA. Lysates were immunoblotted with the indicated antibodies. C–E. BT-549 (C), MDA-MB-231 (D), and BT-20/MUC1-C (E) cells treated with 5 μM CP-2 or 5 μM GO-203 for 12 h were analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for CP-2 (assigned a value of 1) (left). Cell lysates treated with 5 μM CP-2 or 5 μM GO-203 for 48 h were immunoblotted with the indicated antibodies (right).
    Figure Legend Snippet: A. Schema of the MUC1-C subunit with the 58 aa extracellular domain (ED), the 28 aa transmembrane domain (TM), and the sequence of the 72 aa cytoplasmic domain (CD). The MUC1-C cytoplasmic domain contains a CQC motif that is necessary and sufficient for MUC1-C homodimerization and oncogenic function. GO-203 is a cell-penetrating peptide that binds the CQC motif and blocks MUC1-C homodimerization. Highlighted are MUC1-C-induced pathways that confer the activation of ZEB1 and MYC. B. BT-549 cells were transfected with a control or MUC1-C(AQA) vector in which the CQC motif had been mutated to AQA. Lysates were immunoblotted with the indicated antibodies. C–E. BT-549 (C), MDA-MB-231 (D), and BT-20/MUC1-C (E) cells treated with 5 μM CP-2 or 5 μM GO-203 for 12 h were analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for CP-2 (assigned a value of 1) (left). Cell lysates treated with 5 μM CP-2 or 5 μM GO-203 for 48 h were immunoblotted with the indicated antibodies (right).

    Techniques Used: Sequencing, Activation Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR

    A. Schema of the BMI1 promoter region with positioning of the putative MYC binding site at −177 to −182 bp upstream of the transcription start site. The BMI1 promoter-luciferase (Luc) pGL3-BMI1PrWT vector includes the wild-type BMI1 promoter and pGL3-BMI1PrMut contains a mutation in the E-Box sequences (CACGTG has been mutated to CGCGTG)(upper panel). BT-549/tet-MUC1shRNA cells cultured with or without DOX for 5 d were transfected with the pGL3-Basic Luc or pGL3-BMI1PrWT reporter for 48 h and then analyzed for luciferase activity. The results (mean±SD of 3 determinations) are expressed as the relative luciferase activity compared to that obtained with pGL3-Basic Luc (assigned a value of 1)(lower panel). B. BT-549 cells were transfected with the pGL3-Basic Luc or pGL3-BMI1PrWT reporter for 6 h and then treated with 5 μM CP-2 or GO-203 for an additional 42 h. The results (mean±SD of 3 determinations) are expressed as the relative luciferase activity compared to that obtained with CP-2-treated cells (assigned a value of 1). C. BT-549 cells were transfected with the pGL3-Basic Luc, pGL3-BMI1PrWT or pGL3-BMI1PrMut reporter for 48 h and then analyzed for luciferase activity (left). BT-549 cells were transfected with (i) a control vector or one expressing MUC1-C, and (ii) the pGL3-Basic Luc, pGL3-BMI1PrWT or pGL3-BMI1PrMut reporter for 72 h and then analyzed for luciferase activity (right). The results (mean±SD of 3 determinations) are expressed as the relative luciferase activity compared to that obtained with pGL3-Basic Luc (assigned a value of 1). D. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 5 d. MYC mRNA levels were determined by qRT-PCR. The results (mean±SD) are expressed as relative MYC mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1) (left). Lysates were immunoblotted with the indicated antibodies (right). E. BT-549/tet-MYCshRNA cells cultured with or without DOX for 12 h were analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1) (left). Cell lysates treated with DOX for 48 h were immunoblotted with the indicated antibodies (right). F. Soluble chromatin from BT-549/tet-MUC1shRNA cells was precipitated with anti-MYC or a control IgG (left). The final DNA samples were amplified by qPCR with primers for the BMI1 promoter. The results (mean±SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). Soluble chromatin from 549/tet-MUC1shRNA cells cultured with or without DOX for 5 d was precipitated with anti-MYC or a control IgG. The final DNA samples were amplified by qPCR. The results (mean±SEM of three determinations) are expressed as the relative fold enrichment compared to that obtained for control DOX-untreated cells (assigned a value of 1) (right).
    Figure Legend Snippet: A. Schema of the BMI1 promoter region with positioning of the putative MYC binding site at −177 to −182 bp upstream of the transcription start site. The BMI1 promoter-luciferase (Luc) pGL3-BMI1PrWT vector includes the wild-type BMI1 promoter and pGL3-BMI1PrMut contains a mutation in the E-Box sequences (CACGTG has been mutated to CGCGTG)(upper panel). BT-549/tet-MUC1shRNA cells cultured with or without DOX for 5 d were transfected with the pGL3-Basic Luc or pGL3-BMI1PrWT reporter for 48 h and then analyzed for luciferase activity. The results (mean±SD of 3 determinations) are expressed as the relative luciferase activity compared to that obtained with pGL3-Basic Luc (assigned a value of 1)(lower panel). B. BT-549 cells were transfected with the pGL3-Basic Luc or pGL3-BMI1PrWT reporter for 6 h and then treated with 5 μM CP-2 or GO-203 for an additional 42 h. The results (mean±SD of 3 determinations) are expressed as the relative luciferase activity compared to that obtained with CP-2-treated cells (assigned a value of 1). C. BT-549 cells were transfected with the pGL3-Basic Luc, pGL3-BMI1PrWT or pGL3-BMI1PrMut reporter for 48 h and then analyzed for luciferase activity (left). BT-549 cells were transfected with (i) a control vector or one expressing MUC1-C, and (ii) the pGL3-Basic Luc, pGL3-BMI1PrWT or pGL3-BMI1PrMut reporter for 72 h and then analyzed for luciferase activity (right). The results (mean±SD of 3 determinations) are expressed as the relative luciferase activity compared to that obtained with pGL3-Basic Luc (assigned a value of 1). D. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 5 d. MYC mRNA levels were determined by qRT-PCR. The results (mean±SD) are expressed as relative MYC mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1) (left). Lysates were immunoblotted with the indicated antibodies (right). E. BT-549/tet-MYCshRNA cells cultured with or without DOX for 12 h were analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1) (left). Cell lysates treated with DOX for 48 h were immunoblotted with the indicated antibodies (right). F. Soluble chromatin from BT-549/tet-MUC1shRNA cells was precipitated with anti-MYC or a control IgG (left). The final DNA samples were amplified by qPCR with primers for the BMI1 promoter. The results (mean±SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). Soluble chromatin from 549/tet-MUC1shRNA cells cultured with or without DOX for 5 d was precipitated with anti-MYC or a control IgG. The final DNA samples were amplified by qPCR. The results (mean±SEM of three determinations) are expressed as the relative fold enrichment compared to that obtained for control DOX-untreated cells (assigned a value of 1) (right).

    Techniques Used: Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Cell Culture, Transfection, Activity Assay, Expressing, Quantitative RT-PCR, Amplification

    A. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 4 d. Lysates were immunoblotted with the indicated antibodies. B. BT-549 cells were transduced with lentiviral vectors to stably express a control shRNA (CshRNA) or a NF-κB p65 shRNA. Lysates were immunoblotted with the indicated antibodies. C. BT-549 cells were treated with control DMSO vehicle or BAY-11-7085 for 16 h. Lysates were immunoblotted with the indicated antibodies. D. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 4 d. The cells were analyzed for miR-200c levels by qRT-PCR. The results (mean±SD) are expressed as relative miR-200c/RNU48 levels compared to that obtained for control DOX-untreated cells (assigned a value of 1). E and F. BT-549/tet-MUC1shRNA cells cultured with DOX for 7 d were transfected with 12.5 nM anti-miR-200c or a negative control oligonucleotide for 4 d. The cells were then analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for control anti-miR-200c-untreated cells (assigned a value of 1) (E). Lysates were immunoblotted with the indicated antibodies (F).
    Figure Legend Snippet: A. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 4 d. Lysates were immunoblotted with the indicated antibodies. B. BT-549 cells were transduced with lentiviral vectors to stably express a control shRNA (CshRNA) or a NF-κB p65 shRNA. Lysates were immunoblotted with the indicated antibodies. C. BT-549 cells were treated with control DMSO vehicle or BAY-11-7085 for 16 h. Lysates were immunoblotted with the indicated antibodies. D. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 4 d. The cells were analyzed for miR-200c levels by qRT-PCR. The results (mean±SD) are expressed as relative miR-200c/RNU48 levels compared to that obtained for control DOX-untreated cells (assigned a value of 1). E and F. BT-549/tet-MUC1shRNA cells cultured with DOX for 7 d were transfected with 12.5 nM anti-miR-200c or a negative control oligonucleotide for 4 d. The cells were then analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for control anti-miR-200c-untreated cells (assigned a value of 1) (E). Lysates were immunoblotted with the indicated antibodies (F).

    Techniques Used: Transduction, Stable Transfection, shRNA, Quantitative RT-PCR, Cell Culture, Transfection, Negative Control

    A. Schema of the CDKN2A promoter with positioning of the BMI1-response element (BRE) at −423 to −446 and −474 to −480 bp upstream to the transcription start site. B. Soluble chromatin from BT−549 cells was precipitated with anti-BMI1 or a control IgG. C. In the re-ChIP analysis, BMI1 precipitates were released and re-immunoprecipitated with anti-MUC1-C and a control IgG. D. Soluble chromatin from BT-549 cells was precipitated with anti-MUC1-C or a control IgG. The final DNA samples were amplified by qPCR with primers for the CDKN2A promoter. The results (mean±SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). E. GST and GST-BMI1 were incubated with either purified MUC1-CD or MUC1-CD(AQA). The adsorbates were immunoblotted with anti-MUC1-C. Input of the GST proteins was assessed by Coomassie blue staining. F. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 7 d. p16 INK4a mRNA levels were determined by qRT-PCR. The results (mean±SD) are expressed as relative p16 INK4a mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1) (left). Cell lysates cultured with or without DOX for 12 d were immunoblotted with the indicated antibodies (right).
    Figure Legend Snippet: A. Schema of the CDKN2A promoter with positioning of the BMI1-response element (BRE) at −423 to −446 and −474 to −480 bp upstream to the transcription start site. B. Soluble chromatin from BT−549 cells was precipitated with anti-BMI1 or a control IgG. C. In the re-ChIP analysis, BMI1 precipitates were released and re-immunoprecipitated with anti-MUC1-C and a control IgG. D. Soluble chromatin from BT-549 cells was precipitated with anti-MUC1-C or a control IgG. The final DNA samples were amplified by qPCR with primers for the CDKN2A promoter. The results (mean±SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). E. GST and GST-BMI1 were incubated with either purified MUC1-CD or MUC1-CD(AQA). The adsorbates were immunoblotted with anti-MUC1-C. Input of the GST proteins was assessed by Coomassie blue staining. F. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 7 d. p16 INK4a mRNA levels were determined by qRT-PCR. The results (mean±SD) are expressed as relative p16 INK4a mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1) (left). Cell lysates cultured with or without DOX for 12 d were immunoblotted with the indicated antibodies (right).

    Techniques Used: Immunoprecipitation, Amplification, Incubation, Purification, Staining, Quantitative RT-PCR, Cell Culture

    A. Microarray data from Oncomine database are expressed as box plots (25th–75th percentiles) for MUC1 (upper panel) and BMI1 (lower panel) expression in normal breast tissues (n=61) and breast cancer samples (n=389). B–D. MUC1 and BMI1 gene expression data from METABRIC (B; n=1980), TCGA (C; n=528), and GSE41970 (D; n=54) datasets was assessed for correlation using the Pearson’s correlation coefficient, where p<0.05 was considered as statistically significant. E. Schema depicting the proposed effects of MUC1-C on BMI1 expression and function. MUC1-C drives BMI1 expression by (i) inducing MYC and in turn MYC-mediated activation of the BMI1 gene, and (ii) downregulating miR-200c and thereby inhibiting the suppressive effects of miR-200c on BMI1 mRNA. As a result, MUC1-C promotes the ubiquitylation of H2A and repression of HOXC5 and HOXC13 . MUC1-C also interacts directly with BMI1 and contributes to repression of the CDKN2A promoter and suppression of p16 INK4a expression. These findings and the demonstration that MUC1 and BMI1 significantly correlate in breast tumors supports the notion that MUC1-C is, at least in part, responsible for the upregulation of BMI1 in human cancers, which has been linked to tumor promotion by increasing self-renewal capacity, cancer stem-like cells and genomic instability.
    Figure Legend Snippet: A. Microarray data from Oncomine database are expressed as box plots (25th–75th percentiles) for MUC1 (upper panel) and BMI1 (lower panel) expression in normal breast tissues (n=61) and breast cancer samples (n=389). B–D. MUC1 and BMI1 gene expression data from METABRIC (B; n=1980), TCGA (C; n=528), and GSE41970 (D; n=54) datasets was assessed for correlation using the Pearson’s correlation coefficient, where p<0.05 was considered as statistically significant. E. Schema depicting the proposed effects of MUC1-C on BMI1 expression and function. MUC1-C drives BMI1 expression by (i) inducing MYC and in turn MYC-mediated activation of the BMI1 gene, and (ii) downregulating miR-200c and thereby inhibiting the suppressive effects of miR-200c on BMI1 mRNA. As a result, MUC1-C promotes the ubiquitylation of H2A and repression of HOXC5 and HOXC13 . MUC1-C also interacts directly with BMI1 and contributes to repression of the CDKN2A promoter and suppression of p16 INK4a expression. These findings and the demonstration that MUC1 and BMI1 significantly correlate in breast tumors supports the notion that MUC1-C is, at least in part, responsible for the upregulation of BMI1 in human cancers, which has been linked to tumor promotion by increasing self-renewal capacity, cancer stem-like cells and genomic instability.

    Techniques Used: Microarray, Expressing, Activation Assay

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    Cell Signaling Technology Inc knockdown bmi1 gene
    A Representative images of <t>BMI1</t> staining (green bodies). Nuclei are counterstained with DAPi (blue staining). BMI1 protein can accumulate in CAP bodies (left panel) or has uniform punctate distribution (PcG bodies, right panel). Scale bar: 5 μm. B Stacked bar plot representing the proportion of CAP and PcG bodies in ALDH br and ALDH neg SUM159 cells sorted according to their cell-cycle phase (G0/G1, S, and G2/M). C , D Representative images (left panels) of BMI1 costaining (green foci) with γH2AX (red foci) for cells harboring CAP bodies ( C ) or PcG bodies ( D ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and γH2AX fluorescent signals. E Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/γH2AX colocalization. F , G Representative images (left panels) of BMI1 costaining (green foci) with RAD51 (red foci) for cells harboring CAP bodies ( F ) or PcG bodies ( G ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and RAD51 fluorescent signals. H Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/RAD51 colocalization. I Representative images of BMI1 staining (green bodies) in ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated (CTRL). Nuclei are counterstained with DAPi (blue staining). J Stacked bar plot representing the proportion of CAP and PcG bodies in replicative ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated. K Bar plots representing the proportion of γH2AX-positive cells for each cell subpopulation (replicative ALDH br and ALDH neg SUM159 cells) following 5-aza treatment or in untreated conditions (CTRL). Statistical test used is Student’s t -test. Data represent mean ± SD.
    Knockdown Bmi1 Gene, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc bmi1 sirna
    A–C. BT-549 cells were stably transduced to express a tetracycline-inducible control shRNA (tet-CshRNA) (A) or a MUC1 shRNA (tet-MUC1shRNA) (B). Cells treated with 200 ng/ml DOX for 4 d were analyzed for MUC1 and <t>BMI1</t> mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1). Cell lysates treated with 200 ng/ml DOX for 7 d were immunoblotted with the indicated antibodies (C). D. MDA-MB-231/tet-MUC1shRNA cells treated with 200 ng/ml DOX for 4 d were analyzed for MUC1 and BMI1 mRNA levels by qRT-PCR (left). Cell lysates treated with 200 ng/ml DOX for 7 d were immunoblotted with the indicated antibodies (right). E. BT-20 cells stably expressing a control or MUC1-C vector were analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for vector cells (assigned a value of 1) (left). Lysates were immunoblotted with the indicated antibodies (right).
    Bmi1 Sirna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bmi1 sirna/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bmi1 sirna - by Bioz Stars, 2023-02
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    A Representative images of BMI1 staining (green bodies). Nuclei are counterstained with DAPi (blue staining). BMI1 protein can accumulate in CAP bodies (left panel) or has uniform punctate distribution (PcG bodies, right panel). Scale bar: 5 μm. B Stacked bar plot representing the proportion of CAP and PcG bodies in ALDH br and ALDH neg SUM159 cells sorted according to their cell-cycle phase (G0/G1, S, and G2/M). C , D Representative images (left panels) of BMI1 costaining (green foci) with γH2AX (red foci) for cells harboring CAP bodies ( C ) or PcG bodies ( D ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and γH2AX fluorescent signals. E Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/γH2AX colocalization. F , G Representative images (left panels) of BMI1 costaining (green foci) with RAD51 (red foci) for cells harboring CAP bodies ( F ) or PcG bodies ( G ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and RAD51 fluorescent signals. H Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/RAD51 colocalization. I Representative images of BMI1 staining (green bodies) in ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated (CTRL). Nuclei are counterstained with DAPi (blue staining). J Stacked bar plot representing the proportion of CAP and PcG bodies in replicative ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated. K Bar plots representing the proportion of γH2AX-positive cells for each cell subpopulation (replicative ALDH br and ALDH neg SUM159 cells) following 5-aza treatment or in untreated conditions (CTRL). Statistical test used is Student’s t -test. Data represent mean ± SD.

    Journal: Cell Death & Disease

    Article Title: BMI1 nuclear location is critical for RAD51-dependent response to replication stress and drives chemoresistance in breast cancer stem cells

    doi: 10.1038/s41419-022-04538-w

    Figure Lengend Snippet: A Representative images of BMI1 staining (green bodies). Nuclei are counterstained with DAPi (blue staining). BMI1 protein can accumulate in CAP bodies (left panel) or has uniform punctate distribution (PcG bodies, right panel). Scale bar: 5 μm. B Stacked bar plot representing the proportion of CAP and PcG bodies in ALDH br and ALDH neg SUM159 cells sorted according to their cell-cycle phase (G0/G1, S, and G2/M). C , D Representative images (left panels) of BMI1 costaining (green foci) with γH2AX (red foci) for cells harboring CAP bodies ( C ) or PcG bodies ( D ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and γH2AX fluorescent signals. E Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/γH2AX colocalization. F , G Representative images (left panels) of BMI1 costaining (green foci) with RAD51 (red foci) for cells harboring CAP bodies ( F ) or PcG bodies ( G ). Nuclei are counterstained with DAPi (blue staining). The red lines correspond to the line scans. Pearson’s coefficient evaluated the amount of colocalization. Scale bar: 5 μm. On the right panels, line-scan profiles of the relative intensity of BMI1 and RAD51 fluorescent signals. H Bar plots representing the proportion of replicative ALDH br and ALDH neg SUM159 cells with BMI1/RAD51 colocalization. I Representative images of BMI1 staining (green bodies) in ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated (CTRL). Nuclei are counterstained with DAPi (blue staining). J Stacked bar plot representing the proportion of CAP and PcG bodies in replicative ALDH br and ALDH neg SUM159 cells treated with 5-aza or untreated. K Bar plots representing the proportion of γH2AX-positive cells for each cell subpopulation (replicative ALDH br and ALDH neg SUM159 cells) following 5-aza treatment or in untreated conditions (CTRL). Statistical test used is Student’s t -test. Data represent mean ± SD.

    Article Snippet: We used siRNA gene silencing to specially target and knockdown BMI1 gene. siRNA (6442 S, Cell Signaling) was lipoplexed with Lipofectamine RNAiMAX (Life Technologies) in a 96-well culture plate or in a 100 mm tissue-culture dish.

    Techniques: Staining

    A–C. BT-549 cells were stably transduced to express a tetracycline-inducible control shRNA (tet-CshRNA) (A) or a MUC1 shRNA (tet-MUC1shRNA) (B). Cells treated with 200 ng/ml DOX for 4 d were analyzed for MUC1 and BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1). Cell lysates treated with 200 ng/ml DOX for 7 d were immunoblotted with the indicated antibodies (C). D. MDA-MB-231/tet-MUC1shRNA cells treated with 200 ng/ml DOX for 4 d were analyzed for MUC1 and BMI1 mRNA levels by qRT-PCR (left). Cell lysates treated with 200 ng/ml DOX for 7 d were immunoblotted with the indicated antibodies (right). E. BT-20 cells stably expressing a control or MUC1-C vector were analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for vector cells (assigned a value of 1) (left). Lysates were immunoblotted with the indicated antibodies (right).

    Journal: Oncogene

    Article Title: MUC1-C ACTIVATES BMI1 IN HUMAN CANCER CELLS

    doi: 10.1038/onc.2016.439

    Figure Lengend Snippet: A–C. BT-549 cells were stably transduced to express a tetracycline-inducible control shRNA (tet-CshRNA) (A) or a MUC1 shRNA (tet-MUC1shRNA) (B). Cells treated with 200 ng/ml DOX for 4 d were analyzed for MUC1 and BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1). Cell lysates treated with 200 ng/ml DOX for 7 d were immunoblotted with the indicated antibodies (C). D. MDA-MB-231/tet-MUC1shRNA cells treated with 200 ng/ml DOX for 4 d were analyzed for MUC1 and BMI1 mRNA levels by qRT-PCR (left). Cell lysates treated with 200 ng/ml DOX for 7 d were immunoblotted with the indicated antibodies (right). E. BT-20 cells stably expressing a control or MUC1-C vector were analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for vector cells (assigned a value of 1) (left). Lysates were immunoblotted with the indicated antibodies (right).

    Article Snippet: Cells were cultured to 60% to 80% confluence and transiently transfected with (i) anti-miR-200c or a negative control oligonucleotide (Ambion, Carlsbad, CA, USA) and (ii) a BMI1 siRNA or control siRNA (Cell Signaling Technology, Danvers, MA, USA) using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA) in accordance with the protocol provided by the manufacturer.

    Techniques: Stable Transfection, shRNA, Quantitative RT-PCR, Expressing, Plasmid Preparation

    A. Schema of the MUC1-C subunit with the 58 aa extracellular domain (ED), the 28 aa transmembrane domain (TM), and the sequence of the 72 aa cytoplasmic domain (CD). The MUC1-C cytoplasmic domain contains a CQC motif that is necessary and sufficient for MUC1-C homodimerization and oncogenic function. GO-203 is a cell-penetrating peptide that binds the CQC motif and blocks MUC1-C homodimerization. Highlighted are MUC1-C-induced pathways that confer the activation of ZEB1 and MYC. B. BT-549 cells were transfected with a control or MUC1-C(AQA) vector in which the CQC motif had been mutated to AQA. Lysates were immunoblotted with the indicated antibodies. C–E. BT-549 (C), MDA-MB-231 (D), and BT-20/MUC1-C (E) cells treated with 5 μM CP-2 or 5 μM GO-203 for 12 h were analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for CP-2 (assigned a value of 1) (left). Cell lysates treated with 5 μM CP-2 or 5 μM GO-203 for 48 h were immunoblotted with the indicated antibodies (right).

    Journal: Oncogene

    Article Title: MUC1-C ACTIVATES BMI1 IN HUMAN CANCER CELLS

    doi: 10.1038/onc.2016.439

    Figure Lengend Snippet: A. Schema of the MUC1-C subunit with the 58 aa extracellular domain (ED), the 28 aa transmembrane domain (TM), and the sequence of the 72 aa cytoplasmic domain (CD). The MUC1-C cytoplasmic domain contains a CQC motif that is necessary and sufficient for MUC1-C homodimerization and oncogenic function. GO-203 is a cell-penetrating peptide that binds the CQC motif and blocks MUC1-C homodimerization. Highlighted are MUC1-C-induced pathways that confer the activation of ZEB1 and MYC. B. BT-549 cells were transfected with a control or MUC1-C(AQA) vector in which the CQC motif had been mutated to AQA. Lysates were immunoblotted with the indicated antibodies. C–E. BT-549 (C), MDA-MB-231 (D), and BT-20/MUC1-C (E) cells treated with 5 μM CP-2 or 5 μM GO-203 for 12 h were analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for CP-2 (assigned a value of 1) (left). Cell lysates treated with 5 μM CP-2 or 5 μM GO-203 for 48 h were immunoblotted with the indicated antibodies (right).

    Article Snippet: Cells were cultured to 60% to 80% confluence and transiently transfected with (i) anti-miR-200c or a negative control oligonucleotide (Ambion, Carlsbad, CA, USA) and (ii) a BMI1 siRNA or control siRNA (Cell Signaling Technology, Danvers, MA, USA) using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA) in accordance with the protocol provided by the manufacturer.

    Techniques: Sequencing, Activation Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR

    A. Schema of the BMI1 promoter region with positioning of the putative MYC binding site at −177 to −182 bp upstream of the transcription start site. The BMI1 promoter-luciferase (Luc) pGL3-BMI1PrWT vector includes the wild-type BMI1 promoter and pGL3-BMI1PrMut contains a mutation in the E-Box sequences (CACGTG has been mutated to CGCGTG)(upper panel). BT-549/tet-MUC1shRNA cells cultured with or without DOX for 5 d were transfected with the pGL3-Basic Luc or pGL3-BMI1PrWT reporter for 48 h and then analyzed for luciferase activity. The results (mean±SD of 3 determinations) are expressed as the relative luciferase activity compared to that obtained with pGL3-Basic Luc (assigned a value of 1)(lower panel). B. BT-549 cells were transfected with the pGL3-Basic Luc or pGL3-BMI1PrWT reporter for 6 h and then treated with 5 μM CP-2 or GO-203 for an additional 42 h. The results (mean±SD of 3 determinations) are expressed as the relative luciferase activity compared to that obtained with CP-2-treated cells (assigned a value of 1). C. BT-549 cells were transfected with the pGL3-Basic Luc, pGL3-BMI1PrWT or pGL3-BMI1PrMut reporter for 48 h and then analyzed for luciferase activity (left). BT-549 cells were transfected with (i) a control vector or one expressing MUC1-C, and (ii) the pGL3-Basic Luc, pGL3-BMI1PrWT or pGL3-BMI1PrMut reporter for 72 h and then analyzed for luciferase activity (right). The results (mean±SD of 3 determinations) are expressed as the relative luciferase activity compared to that obtained with pGL3-Basic Luc (assigned a value of 1). D. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 5 d. MYC mRNA levels were determined by qRT-PCR. The results (mean±SD) are expressed as relative MYC mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1) (left). Lysates were immunoblotted with the indicated antibodies (right). E. BT-549/tet-MYCshRNA cells cultured with or without DOX for 12 h were analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1) (left). Cell lysates treated with DOX for 48 h were immunoblotted with the indicated antibodies (right). F. Soluble chromatin from BT-549/tet-MUC1shRNA cells was precipitated with anti-MYC or a control IgG (left). The final DNA samples were amplified by qPCR with primers for the BMI1 promoter. The results (mean±SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). Soluble chromatin from 549/tet-MUC1shRNA cells cultured with or without DOX for 5 d was precipitated with anti-MYC or a control IgG. The final DNA samples were amplified by qPCR. The results (mean±SEM of three determinations) are expressed as the relative fold enrichment compared to that obtained for control DOX-untreated cells (assigned a value of 1) (right).

    Journal: Oncogene

    Article Title: MUC1-C ACTIVATES BMI1 IN HUMAN CANCER CELLS

    doi: 10.1038/onc.2016.439

    Figure Lengend Snippet: A. Schema of the BMI1 promoter region with positioning of the putative MYC binding site at −177 to −182 bp upstream of the transcription start site. The BMI1 promoter-luciferase (Luc) pGL3-BMI1PrWT vector includes the wild-type BMI1 promoter and pGL3-BMI1PrMut contains a mutation in the E-Box sequences (CACGTG has been mutated to CGCGTG)(upper panel). BT-549/tet-MUC1shRNA cells cultured with or without DOX for 5 d were transfected with the pGL3-Basic Luc or pGL3-BMI1PrWT reporter for 48 h and then analyzed for luciferase activity. The results (mean±SD of 3 determinations) are expressed as the relative luciferase activity compared to that obtained with pGL3-Basic Luc (assigned a value of 1)(lower panel). B. BT-549 cells were transfected with the pGL3-Basic Luc or pGL3-BMI1PrWT reporter for 6 h and then treated with 5 μM CP-2 or GO-203 for an additional 42 h. The results (mean±SD of 3 determinations) are expressed as the relative luciferase activity compared to that obtained with CP-2-treated cells (assigned a value of 1). C. BT-549 cells were transfected with the pGL3-Basic Luc, pGL3-BMI1PrWT or pGL3-BMI1PrMut reporter for 48 h and then analyzed for luciferase activity (left). BT-549 cells were transfected with (i) a control vector or one expressing MUC1-C, and (ii) the pGL3-Basic Luc, pGL3-BMI1PrWT or pGL3-BMI1PrMut reporter for 72 h and then analyzed for luciferase activity (right). The results (mean±SD of 3 determinations) are expressed as the relative luciferase activity compared to that obtained with pGL3-Basic Luc (assigned a value of 1). D. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 5 d. MYC mRNA levels were determined by qRT-PCR. The results (mean±SD) are expressed as relative MYC mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1) (left). Lysates were immunoblotted with the indicated antibodies (right). E. BT-549/tet-MYCshRNA cells cultured with or without DOX for 12 h were analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1) (left). Cell lysates treated with DOX for 48 h were immunoblotted with the indicated antibodies (right). F. Soluble chromatin from BT-549/tet-MUC1shRNA cells was precipitated with anti-MYC or a control IgG (left). The final DNA samples were amplified by qPCR with primers for the BMI1 promoter. The results (mean±SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). Soluble chromatin from 549/tet-MUC1shRNA cells cultured with or without DOX for 5 d was precipitated with anti-MYC or a control IgG. The final DNA samples were amplified by qPCR. The results (mean±SEM of three determinations) are expressed as the relative fold enrichment compared to that obtained for control DOX-untreated cells (assigned a value of 1) (right).

    Article Snippet: Cells were cultured to 60% to 80% confluence and transiently transfected with (i) anti-miR-200c or a negative control oligonucleotide (Ambion, Carlsbad, CA, USA) and (ii) a BMI1 siRNA or control siRNA (Cell Signaling Technology, Danvers, MA, USA) using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA) in accordance with the protocol provided by the manufacturer.

    Techniques: Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Cell Culture, Transfection, Activity Assay, Expressing, Quantitative RT-PCR, Amplification

    A. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 4 d. Lysates were immunoblotted with the indicated antibodies. B. BT-549 cells were transduced with lentiviral vectors to stably express a control shRNA (CshRNA) or a NF-κB p65 shRNA. Lysates were immunoblotted with the indicated antibodies. C. BT-549 cells were treated with control DMSO vehicle or BAY-11-7085 for 16 h. Lysates were immunoblotted with the indicated antibodies. D. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 4 d. The cells were analyzed for miR-200c levels by qRT-PCR. The results (mean±SD) are expressed as relative miR-200c/RNU48 levels compared to that obtained for control DOX-untreated cells (assigned a value of 1). E and F. BT-549/tet-MUC1shRNA cells cultured with DOX for 7 d were transfected with 12.5 nM anti-miR-200c or a negative control oligonucleotide for 4 d. The cells were then analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for control anti-miR-200c-untreated cells (assigned a value of 1) (E). Lysates were immunoblotted with the indicated antibodies (F).

    Journal: Oncogene

    Article Title: MUC1-C ACTIVATES BMI1 IN HUMAN CANCER CELLS

    doi: 10.1038/onc.2016.439

    Figure Lengend Snippet: A. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 4 d. Lysates were immunoblotted with the indicated antibodies. B. BT-549 cells were transduced with lentiviral vectors to stably express a control shRNA (CshRNA) or a NF-κB p65 shRNA. Lysates were immunoblotted with the indicated antibodies. C. BT-549 cells were treated with control DMSO vehicle or BAY-11-7085 for 16 h. Lysates were immunoblotted with the indicated antibodies. D. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 4 d. The cells were analyzed for miR-200c levels by qRT-PCR. The results (mean±SD) are expressed as relative miR-200c/RNU48 levels compared to that obtained for control DOX-untreated cells (assigned a value of 1). E and F. BT-549/tet-MUC1shRNA cells cultured with DOX for 7 d were transfected with 12.5 nM anti-miR-200c or a negative control oligonucleotide for 4 d. The cells were then analyzed for BMI1 mRNA levels by qRT-PCR. The results (mean±SD) are expressed as relative BMI1 mRNA levels compared to that obtained for control anti-miR-200c-untreated cells (assigned a value of 1) (E). Lysates were immunoblotted with the indicated antibodies (F).

    Article Snippet: Cells were cultured to 60% to 80% confluence and transiently transfected with (i) anti-miR-200c or a negative control oligonucleotide (Ambion, Carlsbad, CA, USA) and (ii) a BMI1 siRNA or control siRNA (Cell Signaling Technology, Danvers, MA, USA) using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA) in accordance with the protocol provided by the manufacturer.

    Techniques: Transduction, Stable Transfection, shRNA, Quantitative RT-PCR, Cell Culture, Transfection, Negative Control

    A. Schema of the CDKN2A promoter with positioning of the BMI1-response element (BRE) at −423 to −446 and −474 to −480 bp upstream to the transcription start site. B. Soluble chromatin from BT−549 cells was precipitated with anti-BMI1 or a control IgG. C. In the re-ChIP analysis, BMI1 precipitates were released and re-immunoprecipitated with anti-MUC1-C and a control IgG. D. Soluble chromatin from BT-549 cells was precipitated with anti-MUC1-C or a control IgG. The final DNA samples were amplified by qPCR with primers for the CDKN2A promoter. The results (mean±SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). E. GST and GST-BMI1 were incubated with either purified MUC1-CD or MUC1-CD(AQA). The adsorbates were immunoblotted with anti-MUC1-C. Input of the GST proteins was assessed by Coomassie blue staining. F. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 7 d. p16 INK4a mRNA levels were determined by qRT-PCR. The results (mean±SD) are expressed as relative p16 INK4a mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1) (left). Cell lysates cultured with or without DOX for 12 d were immunoblotted with the indicated antibodies (right).

    Journal: Oncogene

    Article Title: MUC1-C ACTIVATES BMI1 IN HUMAN CANCER CELLS

    doi: 10.1038/onc.2016.439

    Figure Lengend Snippet: A. Schema of the CDKN2A promoter with positioning of the BMI1-response element (BRE) at −423 to −446 and −474 to −480 bp upstream to the transcription start site. B. Soluble chromatin from BT−549 cells was precipitated with anti-BMI1 or a control IgG. C. In the re-ChIP analysis, BMI1 precipitates were released and re-immunoprecipitated with anti-MUC1-C and a control IgG. D. Soluble chromatin from BT-549 cells was precipitated with anti-MUC1-C or a control IgG. The final DNA samples were amplified by qPCR with primers for the CDKN2A promoter. The results (mean±SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). E. GST and GST-BMI1 were incubated with either purified MUC1-CD or MUC1-CD(AQA). The adsorbates were immunoblotted with anti-MUC1-C. Input of the GST proteins was assessed by Coomassie blue staining. F. BT-549/tet-MUC1shRNA cells were treated with or without DOX for 7 d. p16 INK4a mRNA levels were determined by qRT-PCR. The results (mean±SD) are expressed as relative p16 INK4a mRNA levels compared to that obtained for control DOX-untreated cells (assigned a value of 1) (left). Cell lysates cultured with or without DOX for 12 d were immunoblotted with the indicated antibodies (right).

    Article Snippet: Cells were cultured to 60% to 80% confluence and transiently transfected with (i) anti-miR-200c or a negative control oligonucleotide (Ambion, Carlsbad, CA, USA) and (ii) a BMI1 siRNA or control siRNA (Cell Signaling Technology, Danvers, MA, USA) using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA) in accordance with the protocol provided by the manufacturer.

    Techniques: Immunoprecipitation, Amplification, Incubation, Purification, Staining, Quantitative RT-PCR, Cell Culture

    A. Microarray data from Oncomine database are expressed as box plots (25th–75th percentiles) for MUC1 (upper panel) and BMI1 (lower panel) expression in normal breast tissues (n=61) and breast cancer samples (n=389). B–D. MUC1 and BMI1 gene expression data from METABRIC (B; n=1980), TCGA (C; n=528), and GSE41970 (D; n=54) datasets was assessed for correlation using the Pearson’s correlation coefficient, where p<0.05 was considered as statistically significant. E. Schema depicting the proposed effects of MUC1-C on BMI1 expression and function. MUC1-C drives BMI1 expression by (i) inducing MYC and in turn MYC-mediated activation of the BMI1 gene, and (ii) downregulating miR-200c and thereby inhibiting the suppressive effects of miR-200c on BMI1 mRNA. As a result, MUC1-C promotes the ubiquitylation of H2A and repression of HOXC5 and HOXC13 . MUC1-C also interacts directly with BMI1 and contributes to repression of the CDKN2A promoter and suppression of p16 INK4a expression. These findings and the demonstration that MUC1 and BMI1 significantly correlate in breast tumors supports the notion that MUC1-C is, at least in part, responsible for the upregulation of BMI1 in human cancers, which has been linked to tumor promotion by increasing self-renewal capacity, cancer stem-like cells and genomic instability.

    Journal: Oncogene

    Article Title: MUC1-C ACTIVATES BMI1 IN HUMAN CANCER CELLS

    doi: 10.1038/onc.2016.439

    Figure Lengend Snippet: A. Microarray data from Oncomine database are expressed as box plots (25th–75th percentiles) for MUC1 (upper panel) and BMI1 (lower panel) expression in normal breast tissues (n=61) and breast cancer samples (n=389). B–D. MUC1 and BMI1 gene expression data from METABRIC (B; n=1980), TCGA (C; n=528), and GSE41970 (D; n=54) datasets was assessed for correlation using the Pearson’s correlation coefficient, where p<0.05 was considered as statistically significant. E. Schema depicting the proposed effects of MUC1-C on BMI1 expression and function. MUC1-C drives BMI1 expression by (i) inducing MYC and in turn MYC-mediated activation of the BMI1 gene, and (ii) downregulating miR-200c and thereby inhibiting the suppressive effects of miR-200c on BMI1 mRNA. As a result, MUC1-C promotes the ubiquitylation of H2A and repression of HOXC5 and HOXC13 . MUC1-C also interacts directly with BMI1 and contributes to repression of the CDKN2A promoter and suppression of p16 INK4a expression. These findings and the demonstration that MUC1 and BMI1 significantly correlate in breast tumors supports the notion that MUC1-C is, at least in part, responsible for the upregulation of BMI1 in human cancers, which has been linked to tumor promotion by increasing self-renewal capacity, cancer stem-like cells and genomic instability.

    Article Snippet: Cells were cultured to 60% to 80% confluence and transiently transfected with (i) anti-miR-200c or a negative control oligonucleotide (Ambion, Carlsbad, CA, USA) and (ii) a BMI1 siRNA or control siRNA (Cell Signaling Technology, Danvers, MA, USA) using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA) in accordance with the protocol provided by the manufacturer.

    Techniques: Microarray, Expressing, Activation Assay