human breast cancer cell lines bt 549  (ATCC)


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    ATCC human breast cancer cell lines bt 549
    qRT-PCR analysis for FILIP1L conducted on cDNA from <t>BT-549</t> breast ( A ), HT-29 colon ( B ), H1299 lung ( C ) and MIA PaCa-2 pancreatic ( D ) cancer cell lines treated with either 5-aza-2’-deoxycytidine (DAC) or Trichostatin A (TSA). Values 0.1, 1 or 10 in the x axis indicate the concentration of DAC and TSA in μM. The y axis represents fold change of each reagent-treated cell type over DMSO-treated control cells, where each value was standardized with the housekeeping gene hRPL7 . Error bars indicate SEM ( n = 3). The result is an average of two independent experiments. P values are derived from comparison between DMSO-treated control and either DAC- or TSA-treated experiments. NS indicates not significant.
    Human Breast Cancer Cell Lines Bt 549, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Down-Regulation of Fil amin A i nteracting p rotein 1 - l ike Is Associated with Promoter Methylation and an Invasive Phenotype in Breast, Colon, Lung and Pancreatic Cancers"

    Article Title: Down-Regulation of Fil amin A i nteracting p rotein 1 - l ike Is Associated with Promoter Methylation and an Invasive Phenotype in Breast, Colon, Lung and Pancreatic Cancers

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082620

    qRT-PCR analysis for FILIP1L conducted on cDNA from BT-549 breast ( A ), HT-29 colon ( B ), H1299 lung ( C ) and MIA PaCa-2 pancreatic ( D ) cancer cell lines treated with either 5-aza-2’-deoxycytidine (DAC) or Trichostatin A (TSA). Values 0.1, 1 or 10 in the x axis indicate the concentration of DAC and TSA in μM. The y axis represents fold change of each reagent-treated cell type over DMSO-treated control cells, where each value was standardized with the housekeeping gene hRPL7 . Error bars indicate SEM ( n = 3). The result is an average of two independent experiments. P values are derived from comparison between DMSO-treated control and either DAC- or TSA-treated experiments. NS indicates not significant.
    Figure Legend Snippet: qRT-PCR analysis for FILIP1L conducted on cDNA from BT-549 breast ( A ), HT-29 colon ( B ), H1299 lung ( C ) and MIA PaCa-2 pancreatic ( D ) cancer cell lines treated with either 5-aza-2’-deoxycytidine (DAC) or Trichostatin A (TSA). Values 0.1, 1 or 10 in the x axis indicate the concentration of DAC and TSA in μM. The y axis represents fold change of each reagent-treated cell type over DMSO-treated control cells, where each value was standardized with the housekeeping gene hRPL7 . Error bars indicate SEM ( n = 3). The result is an average of two independent experiments. P values are derived from comparison between DMSO-treated control and either DAC- or TSA-treated experiments. NS indicates not significant.

    Techniques Used: Quantitative RT-PCR, Concentration Assay, Derivative Assay

    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

    human breast cancer cell lines bt 549  (ATCC)


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    ATCC human breast cancer cell lines bt 549
    qRT-PCR analysis for FILIP1L conducted on cDNA from <t>BT-549</t> breast ( A ), HT-29 colon ( B ), H1299 lung ( C ) and MIA PaCa-2 pancreatic ( D ) cancer cell lines treated with either 5-aza-2’-deoxycytidine (DAC) or Trichostatin A (TSA). Values 0.1, 1 or 10 in the x axis indicate the concentration of DAC and TSA in μM. The y axis represents fold change of each reagent-treated cell type over DMSO-treated control cells, where each value was standardized with the housekeeping gene hRPL7 . Error bars indicate SEM ( n = 3). The result is an average of two independent experiments. P values are derived from comparison between DMSO-treated control and either DAC- or TSA-treated experiments. NS indicates not significant.
    Human Breast Cancer Cell Lines Bt 549, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human breast cancer cell lines bt 549/product/ATCC
    Average 96 stars, based on 1 article reviews
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    1) Product Images from "Down-Regulation of Fil amin A i nteracting p rotein 1 - l ike Is Associated with Promoter Methylation and an Invasive Phenotype in Breast, Colon, Lung and Pancreatic Cancers"

    Article Title: Down-Regulation of Fil amin A i nteracting p rotein 1 - l ike Is Associated with Promoter Methylation and an Invasive Phenotype in Breast, Colon, Lung and Pancreatic Cancers

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082620

    qRT-PCR analysis for FILIP1L conducted on cDNA from BT-549 breast ( A ), HT-29 colon ( B ), H1299 lung ( C ) and MIA PaCa-2 pancreatic ( D ) cancer cell lines treated with either 5-aza-2’-deoxycytidine (DAC) or Trichostatin A (TSA). Values 0.1, 1 or 10 in the x axis indicate the concentration of DAC and TSA in μM. The y axis represents fold change of each reagent-treated cell type over DMSO-treated control cells, where each value was standardized with the housekeeping gene hRPL7 . Error bars indicate SEM ( n = 3). The result is an average of two independent experiments. P values are derived from comparison between DMSO-treated control and either DAC- or TSA-treated experiments. NS indicates not significant.
    Figure Legend Snippet: qRT-PCR analysis for FILIP1L conducted on cDNA from BT-549 breast ( A ), HT-29 colon ( B ), H1299 lung ( C ) and MIA PaCa-2 pancreatic ( D ) cancer cell lines treated with either 5-aza-2’-deoxycytidine (DAC) or Trichostatin A (TSA). Values 0.1, 1 or 10 in the x axis indicate the concentration of DAC and TSA in μM. The y axis represents fold change of each reagent-treated cell type over DMSO-treated control cells, where each value was standardized with the housekeeping gene hRPL7 . Error bars indicate SEM ( n = 3). The result is an average of two independent experiments. P values are derived from comparison between DMSO-treated control and either DAC- or TSA-treated experiments. NS indicates not significant.

    Techniques Used: Quantitative RT-PCR, Concentration Assay, Derivative Assay

    human basal like breast cancer cell lines  (ATCC)


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    ATCC human basal like breast cancer cell lines
    Human Basal Like Breast Cancer Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, 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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    Structured Review

    ATCC human breast cancer cell line bt 549
    C4ST-1 deficiency suppresses the proliferation of <t>BT-549</t> cells. (A) Growth curves of parental BT-549 and C4ST-1 KO cells. (B) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) was measured by CytoTox-ONE TM Assay. (C) Proliferation of BT-549 and C4ST-1 KO cells was examined by colony formation assay. Both the cell types were seeded at a concentration of 50 cells/well in 6-well plate and cultured for 9 days. The colonies were stained with crystal violet, and observed under a light microscope (Left). The number of colonies of BT-549 ( n = 3) and C4ST-1 KO cells ( n = 3) was compared (Right). (D) The level of cyclin D1 in BT-549 and C4ST-1 KO cells was measured by real-time PCR ( n = 3 each). (E) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without Chase ABC was examined by CytoTox-ONE TM Assay. Cells were digested in the serum-free medium ASF Medium 104 for 4 days by adding 5 munits/well of Chase ABC twice at 0 and 2 days. (F) Cells digested with or without Chase ABC were stained by anti-CS antibody (clone 2B6), which detects the terminal unsaturated disaccharide of CS chains generated by Chase ABC. (G) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without GM6001 was measured. Statistical significance was determined using Student’s t -test. Statistical analyses were performed using KaleidaGraph version 4.5.1.
    Human Breast Cancer Cell Line Bt 549, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation"

    Article Title: Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2021.659428

    C4ST-1 deficiency suppresses the proliferation of BT-549 cells. (A) Growth curves of parental BT-549 and C4ST-1 KO cells. (B) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) was measured by CytoTox-ONE TM Assay. (C) Proliferation of BT-549 and C4ST-1 KO cells was examined by colony formation assay. Both the cell types were seeded at a concentration of 50 cells/well in 6-well plate and cultured for 9 days. The colonies were stained with crystal violet, and observed under a light microscope (Left). The number of colonies of BT-549 ( n = 3) and C4ST-1 KO cells ( n = 3) was compared (Right). (D) The level of cyclin D1 in BT-549 and C4ST-1 KO cells was measured by real-time PCR ( n = 3 each). (E) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without Chase ABC was examined by CytoTox-ONE TM Assay. Cells were digested in the serum-free medium ASF Medium 104 for 4 days by adding 5 munits/well of Chase ABC twice at 0 and 2 days. (F) Cells digested with or without Chase ABC were stained by anti-CS antibody (clone 2B6), which detects the terminal unsaturated disaccharide of CS chains generated by Chase ABC. (G) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without GM6001 was measured. Statistical significance was determined using Student’s t -test. Statistical analyses were performed using KaleidaGraph version 4.5.1.
    Figure Legend Snippet: C4ST-1 deficiency suppresses the proliferation of BT-549 cells. (A) Growth curves of parental BT-549 and C4ST-1 KO cells. (B) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) was measured by CytoTox-ONE TM Assay. (C) Proliferation of BT-549 and C4ST-1 KO cells was examined by colony formation assay. Both the cell types were seeded at a concentration of 50 cells/well in 6-well plate and cultured for 9 days. The colonies were stained with crystal violet, and observed under a light microscope (Left). The number of colonies of BT-549 ( n = 3) and C4ST-1 KO cells ( n = 3) was compared (Right). (D) The level of cyclin D1 in BT-549 and C4ST-1 KO cells was measured by real-time PCR ( n = 3 each). (E) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without Chase ABC was examined by CytoTox-ONE TM Assay. Cells were digested in the serum-free medium ASF Medium 104 for 4 days by adding 5 munits/well of Chase ABC twice at 0 and 2 days. (F) Cells digested with or without Chase ABC were stained by anti-CS antibody (clone 2B6), which detects the terminal unsaturated disaccharide of CS chains generated by Chase ABC. (G) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without GM6001 was measured. Statistical significance was determined using Student’s t -test. Statistical analyses were performed using KaleidaGraph version 4.5.1.

    Techniques Used: Colony Assay, Concentration Assay, Cell Culture, Staining, Light Microscopy, Real-time Polymerase Chain Reaction, Generated

    Inhibition of the cleavage of SDC1 by loss of C4ST-1 expression. (A) Schematic representation of the cleavage sites of MMPs, HS/CS attachment sites on SDC1, and the epitope of the anti-SDC1 antibody used in this study. CS chains are indicated by dashed lines because Ser 206 and Ser 216 are not modified with CS chains in BT-549 cells. In addition, CS chains are attached to Ser 37 , Ser 45 , or Ser 47 , because a SDC1 core protein is modified with CS chains according to our previous study . Anti-SDC1 antibody used in this study recognizes the SDC1 fragment (approx. 32.5 kDa) after cleavage by MMPs. (B) BT-549 and C4ST-1 KO cells were incubated in the presence (+) or absence (−) of GM6001. Cell lysates and conditioned medium prepared from each cell were treated with (+) or without (−) Chase ABC, HSase, and Hepase, and then subject to immunoblotting using the anti-SDC1 antibody. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. (C) The surface expression of SDC1 in BT-549 and BT-549 treated with GM6001, and C4ST-1KO cells were examined by flow cytometry. (D) The expression level of MMP2, MMP9, and MMP14 in BT-549 and C4ST-1KO cells was examined by immunoblotting. (E) The levels of MMP2, MMP7, MMP9, and MMP14 in BT-549 cells transfected either with si-Control or si-MMP2, 7, 9, or 14 were measured by real-time PCR. (F) The effect of knockdown of MMP2, 7, 9, or 14 on the cleavage of SDC1 was examined. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. At the right side, the ratio of cleaved SDC1 to full-length SDC1 is shown as fold change relative to that of siControl. (G) The effect of knockdown of MMP2, MMP7, MMP9, and MMP14 on the proliferation of BT-549 cells ( n = 4, each) was examined by CytoTox-ONE TM Assay. (H) The effect of knockdown of β-catenin on the proliferation of BT-549 cells ( n = 6) was investigated. Statistical significance was determined using Student’s t -test.
    Figure Legend Snippet: Inhibition of the cleavage of SDC1 by loss of C4ST-1 expression. (A) Schematic representation of the cleavage sites of MMPs, HS/CS attachment sites on SDC1, and the epitope of the anti-SDC1 antibody used in this study. CS chains are indicated by dashed lines because Ser 206 and Ser 216 are not modified with CS chains in BT-549 cells. In addition, CS chains are attached to Ser 37 , Ser 45 , or Ser 47 , because a SDC1 core protein is modified with CS chains according to our previous study . Anti-SDC1 antibody used in this study recognizes the SDC1 fragment (approx. 32.5 kDa) after cleavage by MMPs. (B) BT-549 and C4ST-1 KO cells were incubated in the presence (+) or absence (−) of GM6001. Cell lysates and conditioned medium prepared from each cell were treated with (+) or without (−) Chase ABC, HSase, and Hepase, and then subject to immunoblotting using the anti-SDC1 antibody. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. (C) The surface expression of SDC1 in BT-549 and BT-549 treated with GM6001, and C4ST-1KO cells were examined by flow cytometry. (D) The expression level of MMP2, MMP9, and MMP14 in BT-549 and C4ST-1KO cells was examined by immunoblotting. (E) The levels of MMP2, MMP7, MMP9, and MMP14 in BT-549 cells transfected either with si-Control or si-MMP2, 7, 9, or 14 were measured by real-time PCR. (F) The effect of knockdown of MMP2, 7, 9, or 14 on the cleavage of SDC1 was examined. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. At the right side, the ratio of cleaved SDC1 to full-length SDC1 is shown as fold change relative to that of siControl. (G) The effect of knockdown of MMP2, MMP7, MMP9, and MMP14 on the proliferation of BT-549 cells ( n = 4, each) was examined by CytoTox-ONE TM Assay. (H) The effect of knockdown of β-catenin on the proliferation of BT-549 cells ( n = 6) was investigated. Statistical significance was determined using Student’s t -test.

    Techniques Used: Inhibition, Expressing, Modification, Incubation, Western Blot, Flow Cytometry, Transfection, Real-time Polymerase Chain Reaction

    Suppression of SUMOylation of AKT1 by the loss of C4ST-1 expression. (A) Some of the signaling pathways involved in cancer proliferation examined in this study are shown. (B) Phosphorylation of PI3K, AKT1, S6K, ERK1/2, and STAT3 in BT-549 and C4ST-1 KO cells was examined by immunoblotting using phospho-specific antibodies and total antibodies. (C) Phosphorylation of PI3K ( n = 3), AKT1 ( n = 5), S6K ( n = 4), ERK1/2 ( n = 4), and STAT3 ( n = 4) in BT-549 and C4ST-1 KO cells was quantified by calculating the ratios of phosphorylated to total protein. (D) The effect of tannic acid and AKT inhibitor (GSK690693) on the proliferation of BT-549 and C4ST-1KO cells. (E) SUMOylation of AKT1 in BT-549 and C4ST-1 KO cells were examined. Both the cells were treated with (+) or without (−) GM6001, and lysed in the absence (−) or presence (+) of N -ethylmaleimide (NEM), which inhibits SUMO proteases. Immunoprecipitated phospho-AKT1 proteins were subject to immunoblotting using anti-pAkt1 and anti-SUMO1 antibodies.
    Figure Legend Snippet: Suppression of SUMOylation of AKT1 by the loss of C4ST-1 expression. (A) Some of the signaling pathways involved in cancer proliferation examined in this study are shown. (B) Phosphorylation of PI3K, AKT1, S6K, ERK1/2, and STAT3 in BT-549 and C4ST-1 KO cells was examined by immunoblotting using phospho-specific antibodies and total antibodies. (C) Phosphorylation of PI3K ( n = 3), AKT1 ( n = 5), S6K ( n = 4), ERK1/2 ( n = 4), and STAT3 ( n = 4) in BT-549 and C4ST-1 KO cells was quantified by calculating the ratios of phosphorylated to total protein. (D) The effect of tannic acid and AKT inhibitor (GSK690693) on the proliferation of BT-549 and C4ST-1KO cells. (E) SUMOylation of AKT1 in BT-549 and C4ST-1 KO cells were examined. Both the cells were treated with (+) or without (−) GM6001, and lysed in the absence (−) or presence (+) of N -ethylmaleimide (NEM), which inhibits SUMO proteases. Immunoprecipitated phospho-AKT1 proteins were subject to immunoblotting using anti-pAkt1 and anti-SUMO1 antibodies.

    Techniques Used: Expressing, Western Blot, Immunoprecipitation

    Cellular localization of SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG and the effect of SDC1 fragments on cell proliferation after cleavage of MMPs. (A) SDC1(FL)-3xFLAG, SDC1(NTF)-3xFLAG, and SDC1(CTF)-3xFLAG were schematically illustrated. Recognition sites of anti-SDC1 antibodies used in this study are shown. (B) The Expression of SDC1(NTF)-3xFLAG was confirmed by immunoblotting. Conditioned medium and cell lysate was digested with (+) or without (−) GAGase (the mixture of Chase ABC, HSase, and Hepase). SDC1(NTF)-3xFLAG was detected in medium digested with GAGase. (C) The expression of SDC1(FL)-3xFLAG or SDC1(CTF)-3xFLAG in stable clones of BT-549 cells overexpressing SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was confirmed by immunoblotting. Each cell lysate was digested with (+) or without (−) GAGase (the mixture of chondroitinase ABC, heparitinase, and heparinase). SDC1(FL)-3xFLAG modified with GAG chains is represented by “SDC1(FL) + GAG.” BT-549 cells stably expressing the empty vector p3xFLAG-CMV14 is represented as “empty.” (D) Expression pattern of exogenously expressed SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was compared with that of endogenous SDC1 by immunofluorescence method using anti-SDC1 antibodies (HPA00618 and D4Y7H) and anti-FLAG antibody. (E) Proliferation of BT-549 cells overexpressing the empty vector ( n = 4) and SDC1(NTF)-3xFLAG ( n = 3), or proliferation of BT-549 cells overexpressing the empty vector ( n = 5), SDC1(FL)-3xFLAG ( n = 5), and SDC1(CTF)-3xFLAG ( n = 5) was measured.
    Figure Legend Snippet: Cellular localization of SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG and the effect of SDC1 fragments on cell proliferation after cleavage of MMPs. (A) SDC1(FL)-3xFLAG, SDC1(NTF)-3xFLAG, and SDC1(CTF)-3xFLAG were schematically illustrated. Recognition sites of anti-SDC1 antibodies used in this study are shown. (B) The Expression of SDC1(NTF)-3xFLAG was confirmed by immunoblotting. Conditioned medium and cell lysate was digested with (+) or without (−) GAGase (the mixture of Chase ABC, HSase, and Hepase). SDC1(NTF)-3xFLAG was detected in medium digested with GAGase. (C) The expression of SDC1(FL)-3xFLAG or SDC1(CTF)-3xFLAG in stable clones of BT-549 cells overexpressing SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was confirmed by immunoblotting. Each cell lysate was digested with (+) or without (−) GAGase (the mixture of chondroitinase ABC, heparitinase, and heparinase). SDC1(FL)-3xFLAG modified with GAG chains is represented by “SDC1(FL) + GAG.” BT-549 cells stably expressing the empty vector p3xFLAG-CMV14 is represented as “empty.” (D) Expression pattern of exogenously expressed SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was compared with that of endogenous SDC1 by immunofluorescence method using anti-SDC1 antibodies (HPA00618 and D4Y7H) and anti-FLAG antibody. (E) Proliferation of BT-549 cells overexpressing the empty vector ( n = 4) and SDC1(NTF)-3xFLAG ( n = 3), or proliferation of BT-549 cells overexpressing the empty vector ( n = 5), SDC1(FL)-3xFLAG ( n = 5), and SDC1(CTF)-3xFLAG ( n = 5) was measured.

    Techniques Used: Expressing, Western Blot, Clone Assay, Modification, Stable Transfection, Plasmid Preparation, Immunofluorescence

    Enhancement of cell proliferation through the SUMOylation of AKT1 upregulated by the expression of the C-terminal fragment of SDC1. (A) The effect of GM6001 on the proliferation of BT-549 cells overexpressing the empty vector ( n = 5), SDC1(FL)-3xFLAG ( n = 5), and SDC1(CTF)-3xFLAG ( n = 5) was examined. (B) The effect of tannic acid on the proliferation of BT-549 cells overexpressing the empty vector ( n = 4), SDC1(FL)-3xFLAG ( n = 4), and SDC1(CTF)-3xFLAG ( n = 4) was examined. (C) SUMOylated proteins in BT-549 cells overexpressing the empty vector and SDC1(CTF)-3xFLAG were captured using the SUMO-QAPTURE-T kit, and subject to immunoblotting using the anti-Akt1 and anti-SUMO1 antibodies.
    Figure Legend Snippet: Enhancement of cell proliferation through the SUMOylation of AKT1 upregulated by the expression of the C-terminal fragment of SDC1. (A) The effect of GM6001 on the proliferation of BT-549 cells overexpressing the empty vector ( n = 5), SDC1(FL)-3xFLAG ( n = 5), and SDC1(CTF)-3xFLAG ( n = 5) was examined. (B) The effect of tannic acid on the proliferation of BT-549 cells overexpressing the empty vector ( n = 4), SDC1(FL)-3xFLAG ( n = 4), and SDC1(CTF)-3xFLAG ( n = 4) was examined. (C) SUMOylated proteins in BT-549 cells overexpressing the empty vector and SDC1(CTF)-3xFLAG were captured using the SUMO-QAPTURE-T kit, and subject to immunoblotting using the anti-Akt1 and anti-SUMO1 antibodies.

    Techniques Used: Expressing, Plasmid Preparation, Western Blot

    C4ST-1 controls BT-549 cell proliferation by regulating the cleavage of SDC1 by MMPs. SDC1 expressed at the cell surface is cleaved by MMPs, and the N-terminal and the C-terminal fragments of SDC1 are generated. Both fragments, SDC1(NTF) and SDC1(CTF), have a positive effect on cell proliferation, and cell proliferation is more affected by SDC1(CTF) than by SDC1(NTF). SDC1(CTF) is involved in SUMOylation of AKT1, although the mechanism underlying SUMOylation by SDC1(CTF) remains unclear. SUMOylation of AKT1 enhances proliferation through S6 kinase signaling pathway. In C4ST-1KO cells, the cleavage of SDC1 by MMPs is suppressed because of reduced expression of MMPs. SUMOylation of AKT1 is inhibited because of decreased SDC1(CTF), and proliferation is slowed down.
    Figure Legend Snippet: C4ST-1 controls BT-549 cell proliferation by regulating the cleavage of SDC1 by MMPs. SDC1 expressed at the cell surface is cleaved by MMPs, and the N-terminal and the C-terminal fragments of SDC1 are generated. Both fragments, SDC1(NTF) and SDC1(CTF), have a positive effect on cell proliferation, and cell proliferation is more affected by SDC1(CTF) than by SDC1(NTF). SDC1(CTF) is involved in SUMOylation of AKT1, although the mechanism underlying SUMOylation by SDC1(CTF) remains unclear. SUMOylation of AKT1 enhances proliferation through S6 kinase signaling pathway. In C4ST-1KO cells, the cleavage of SDC1 by MMPs is suppressed because of reduced expression of MMPs. SUMOylation of AKT1 is inhibited because of decreased SDC1(CTF), and proliferation is slowed down.

    Techniques Used: Generated, Expressing

    human breast cancer cell lines bt 549  (ATCC)


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    Structured Review

    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: 96/100, 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
    a Expression of the indicated MALAT1 transcripts in 1157 single cells derived from the extinction (KTN126, KTN129, and KTN206) and 592 single cells derived from the persistence (KTN102, KTN132, and KTN615) TNBC patients. Data are presented as a scatter plot with p values indicated on each plot (two-tailed t -test). b Strategy to knockdown MALAT1 promoter using dual guide RNA and Cas9 protein. c Successful deletion of the MALAT1 promoter using CRISPR/Cas9 in MDA-MB-231 and <t>BT-549</t> TNBC model. QRT-PCR for MALAT1 expression in BT-549 wt and MALAT1-KO cell models. Data are presented as mean ± SD, n = 3. ** p < 0.005, *** p < 0.0005. d Sanger sequencing confirming intended homozygous deletion of MALAT1 promoter in MDA-MB-231 cells. e Clonogenic assay for BT-549 and BT-549-MALAT1-KO in the presence of different concentration of Paclitaxel or Doxorubicin. f Quantification of CFU data from ( e ). Data are presented as mean ± SD, n = 4. *** p < 0.0005.
    Human Breast Cancer Cell Lines Bt 549, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Single-cell long noncoding RNA (lncRNA) transcriptome implicates MALAT1 in triple-negative breast cancer (TNBC) resistance to neoadjuvant chemotherapy"

    Article Title: Single-cell long noncoding RNA (lncRNA) transcriptome implicates MALAT1 in triple-negative breast cancer (TNBC) resistance to neoadjuvant chemotherapy

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-020-00383-y

    a Expression of the indicated MALAT1 transcripts in 1157 single cells derived from the extinction (KTN126, KTN129, and KTN206) and 592 single cells derived from the persistence (KTN102, KTN132, and KTN615) TNBC patients. Data are presented as a scatter plot with p values indicated on each plot (two-tailed t -test). b Strategy to knockdown MALAT1 promoter using dual guide RNA and Cas9 protein. c Successful deletion of the MALAT1 promoter using CRISPR/Cas9 in MDA-MB-231 and BT-549 TNBC model. QRT-PCR for MALAT1 expression in BT-549 wt and MALAT1-KO cell models. Data are presented as mean ± SD, n = 3. ** p < 0.005, *** p < 0.0005. d Sanger sequencing confirming intended homozygous deletion of MALAT1 promoter in MDA-MB-231 cells. e Clonogenic assay for BT-549 and BT-549-MALAT1-KO in the presence of different concentration of Paclitaxel or Doxorubicin. f Quantification of CFU data from ( e ). Data are presented as mean ± SD, n = 4. *** p < 0.0005.
    Figure Legend Snippet: a Expression of the indicated MALAT1 transcripts in 1157 single cells derived from the extinction (KTN126, KTN129, and KTN206) and 592 single cells derived from the persistence (KTN102, KTN132, and KTN615) TNBC patients. Data are presented as a scatter plot with p values indicated on each plot (two-tailed t -test). b Strategy to knockdown MALAT1 promoter using dual guide RNA and Cas9 protein. c Successful deletion of the MALAT1 promoter using CRISPR/Cas9 in MDA-MB-231 and BT-549 TNBC model. QRT-PCR for MALAT1 expression in BT-549 wt and MALAT1-KO cell models. Data are presented as mean ± SD, n = 3. ** p < 0.005, *** p < 0.0005. d Sanger sequencing confirming intended homozygous deletion of MALAT1 promoter in MDA-MB-231 cells. e Clonogenic assay for BT-549 and BT-549-MALAT1-KO in the presence of different concentration of Paclitaxel or Doxorubicin. f Quantification of CFU data from ( e ). Data are presented as mean ± SD, n = 4. *** p < 0.0005.

    Techniques Used: Expressing, Derivative Assay, Two Tailed Test, CRISPR, Quantitative RT-PCR, Sequencing, Clonogenic Assay, Concentration Assay

    a Heatmap depicting the expression of upregulated (32) and downregulated (129) genes (1.5 ≤ fc ≥ 1.5) in the BT-549 and MDA-MB-231 TNBC models using whole transcriptome analysis. b Principal component analysis (PCA) illustrating the segregation of BT-549, BT-549-MALAT1-KO, MDA-MB-231, and MDA-MB-231-MALAT1-KO based on PC1 and PC2. Ingenuity pathway analysis (IPA) on the 161 common differentially expressed transcripts in MALAT1-KO TNBC models highlighting suppression of INFG, NFKB, and TNF networks ( c ). Suppression of NUPR1, STAT1, RELA, and SREBF1 transcription regulator ( d ), angiogenesis functional category ( e ), as well as oxidative phosphorylation canonical pathway ( f ) in MALAT1-KO TNBC cells.
    Figure Legend Snippet: a Heatmap depicting the expression of upregulated (32) and downregulated (129) genes (1.5 ≤ fc ≥ 1.5) in the BT-549 and MDA-MB-231 TNBC models using whole transcriptome analysis. b Principal component analysis (PCA) illustrating the segregation of BT-549, BT-549-MALAT1-KO, MDA-MB-231, and MDA-MB-231-MALAT1-KO based on PC1 and PC2. Ingenuity pathway analysis (IPA) on the 161 common differentially expressed transcripts in MALAT1-KO TNBC models highlighting suppression of INFG, NFKB, and TNF networks ( c ). Suppression of NUPR1, STAT1, RELA, and SREBF1 transcription regulator ( d ), angiogenesis functional category ( e ), as well as oxidative phosphorylation canonical pathway ( f ) in MALAT1-KO TNBC cells.

    Techniques Used: Expressing, Functional Assay

    BT-549-MALAT1-KO, MDA-MB-231-MALAT1-KO, and corresponding parental lines were subjected to whole transcriptome and gencode v33 lncRNA analysis. a Hierarchical clustering of BT-549-MALAT1-KO, MDA-MB-231-MALAT1-KO and corresponding parental TNBC models based on lncRNA transcriptome. Each column represents one cell and each row represents lncRNA transcript. The expression level of each transcript (log2) in each sample is depicted according to the color scale. b Principal component analysis (PCA) illustrating the segregation of BT-549, BT-549-MALAT1-KO, MDA-MB-231, and MDA-MB-231-MALAT1-KO based on PC1 and PC2. c Heatmap depicting the most significant lncRNAs associated with the MALAT-KO vs parental phenotype. Expression is depicted according to the color scale.
    Figure Legend Snippet: BT-549-MALAT1-KO, MDA-MB-231-MALAT1-KO, and corresponding parental lines were subjected to whole transcriptome and gencode v33 lncRNA analysis. a Hierarchical clustering of BT-549-MALAT1-KO, MDA-MB-231-MALAT1-KO and corresponding parental TNBC models based on lncRNA transcriptome. Each column represents one cell and each row represents lncRNA transcript. The expression level of each transcript (log2) in each sample is depicted according to the color scale. b Principal component analysis (PCA) illustrating the segregation of BT-549, BT-549-MALAT1-KO, MDA-MB-231, and MDA-MB-231-MALAT1-KO based on PC1 and PC2. c Heatmap depicting the most significant lncRNAs associated with the MALAT-KO vs parental phenotype. Expression is depicted according to the color scale.

    Techniques Used: Expressing

    human breast cancer cell line bt 549  (ATCC)


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    ATCC human breast cancer cell line bt 549
    miRNAs negatively regulated HHEX and influenced the migration, invasion, and proliferation ability in MDA-MB-231 and <t>BT-549.</t> (A) HHEX expression of MDA-MB-231 and BT-549 transfected with and without miRNA inhibitors, mimics. (a) HHEX expression in MDA-MB-231. (b) HHEX expression in BT-549. (c) Histogram showed the quantitative result of the HHEX expression in MDA-MB-231 with different treatments. (d) Histogram showed the quantitative result of the HHEX expression in BT-549 with different treatments. (B) Plate clone formation experiment was used to compare the proliferation ability of MDA-MB-231 transfected with and without miRNA inhibitors, mimics. (C) Plate clone formation experiment was used to compare the proliferation ability of BT-549 transfected with and without miRNA inhibitors, mimics. (D) Transwell migration assay showed the cells migration capacity of MDA-MB-231 transfected with and without miRNA inhibitors, mimics (100×). (E) Transwell migration assay showed the cells migration capacity of BT-549 transfected with and without miRNA inhibitors, mimics (100×). (F) Transwell invasion assay of MDA-MB-231 cells transfected with and without miRNA inhibitors, mimics (100×). (G) Transwell invasion assay of BT-549 cells transfected with and without miRNA inhibitors, mimics (100×). NC, normal control; M1, miR-130b mimics; M2, miR-30e mimics; M3, miR-301b mimics; I1, miR-130b inhibitor; I2, miR-30e inhibitors; I3, miR-301b inhibitor.
    Human Breast Cancer Cell Line Bt 549, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Clinicopathological Significances of Cancer Stem Cell-Associated HHEX Expression in Breast Cancer"

    Article Title: Clinicopathological Significances of Cancer Stem Cell-Associated HHEX Expression in Breast Cancer

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2020.605744

    miRNAs negatively regulated HHEX and influenced the migration, invasion, and proliferation ability in MDA-MB-231 and BT-549. (A) HHEX expression of MDA-MB-231 and BT-549 transfected with and without miRNA inhibitors, mimics. (a) HHEX expression in MDA-MB-231. (b) HHEX expression in BT-549. (c) Histogram showed the quantitative result of the HHEX expression in MDA-MB-231 with different treatments. (d) Histogram showed the quantitative result of the HHEX expression in BT-549 with different treatments. (B) Plate clone formation experiment was used to compare the proliferation ability of MDA-MB-231 transfected with and without miRNA inhibitors, mimics. (C) Plate clone formation experiment was used to compare the proliferation ability of BT-549 transfected with and without miRNA inhibitors, mimics. (D) Transwell migration assay showed the cells migration capacity of MDA-MB-231 transfected with and without miRNA inhibitors, mimics (100×). (E) Transwell migration assay showed the cells migration capacity of BT-549 transfected with and without miRNA inhibitors, mimics (100×). (F) Transwell invasion assay of MDA-MB-231 cells transfected with and without miRNA inhibitors, mimics (100×). (G) Transwell invasion assay of BT-549 cells transfected with and without miRNA inhibitors, mimics (100×). NC, normal control; M1, miR-130b mimics; M2, miR-30e mimics; M3, miR-301b mimics; I1, miR-130b inhibitor; I2, miR-30e inhibitors; I3, miR-301b inhibitor.
    Figure Legend Snippet: miRNAs negatively regulated HHEX and influenced the migration, invasion, and proliferation ability in MDA-MB-231 and BT-549. (A) HHEX expression of MDA-MB-231 and BT-549 transfected with and without miRNA inhibitors, mimics. (a) HHEX expression in MDA-MB-231. (b) HHEX expression in BT-549. (c) Histogram showed the quantitative result of the HHEX expression in MDA-MB-231 with different treatments. (d) Histogram showed the quantitative result of the HHEX expression in BT-549 with different treatments. (B) Plate clone formation experiment was used to compare the proliferation ability of MDA-MB-231 transfected with and without miRNA inhibitors, mimics. (C) Plate clone formation experiment was used to compare the proliferation ability of BT-549 transfected with and without miRNA inhibitors, mimics. (D) Transwell migration assay showed the cells migration capacity of MDA-MB-231 transfected with and without miRNA inhibitors, mimics (100×). (E) Transwell migration assay showed the cells migration capacity of BT-549 transfected with and without miRNA inhibitors, mimics (100×). (F) Transwell invasion assay of MDA-MB-231 cells transfected with and without miRNA inhibitors, mimics (100×). (G) Transwell invasion assay of BT-549 cells transfected with and without miRNA inhibitors, mimics (100×). NC, normal control; M1, miR-130b mimics; M2, miR-30e mimics; M3, miR-301b mimics; I1, miR-130b inhibitor; I2, miR-30e inhibitors; I3, miR-301b inhibitor.

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

    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: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human breast cancer cell line bt549 cells  (ATCC)


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    ATCC human breast cancer cell line bt549 cells
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    ATCC human breast cancer cell lines bt 549
    qRT-PCR analysis for FILIP1L conducted on cDNA from <t>BT-549</t> breast ( A ), HT-29 colon ( B ), H1299 lung ( C ) and MIA PaCa-2 pancreatic ( D ) cancer cell lines treated with either 5-aza-2’-deoxycytidine (DAC) or Trichostatin A (TSA). Values 0.1, 1 or 10 in the x axis indicate the concentration of DAC and TSA in μM. The y axis represents fold change of each reagent-treated cell type over DMSO-treated control cells, where each value was standardized with the housekeeping gene hRPL7 . Error bars indicate SEM ( n = 3). The result is an average of two independent experiments. P values are derived from comparison between DMSO-treated control and either DAC- or TSA-treated experiments. NS indicates not significant.
    Human Breast Cancer Cell Lines Bt 549, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    ATCC human basal like breast cancer cell lines
    qRT-PCR analysis for FILIP1L conducted on cDNA from <t>BT-549</t> breast ( A ), HT-29 colon ( B ), H1299 lung ( C ) and MIA PaCa-2 pancreatic ( D ) cancer cell lines treated with either 5-aza-2’-deoxycytidine (DAC) or Trichostatin A (TSA). Values 0.1, 1 or 10 in the x axis indicate the concentration of DAC and TSA in μM. The y axis represents fold change of each reagent-treated cell type over DMSO-treated control cells, where each value was standardized with the housekeeping gene hRPL7 . Error bars indicate SEM ( n = 3). The result is an average of two independent experiments. P values are derived from comparison between DMSO-treated control and either DAC- or TSA-treated experiments. NS indicates not significant.
    Human Basal Like Breast Cancer Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human breast cancer cell line bt 549
    C4ST-1 deficiency suppresses the proliferation of <t>BT-549</t> cells. (A) Growth curves of parental BT-549 and C4ST-1 KO cells. (B) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) was measured by CytoTox-ONE TM Assay. (C) Proliferation of BT-549 and C4ST-1 KO cells was examined by colony formation assay. Both the cell types were seeded at a concentration of 50 cells/well in 6-well plate and cultured for 9 days. The colonies were stained with crystal violet, and observed under a light microscope (Left). The number of colonies of BT-549 ( n = 3) and C4ST-1 KO cells ( n = 3) was compared (Right). (D) The level of cyclin D1 in BT-549 and C4ST-1 KO cells was measured by real-time PCR ( n = 3 each). (E) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without Chase ABC was examined by CytoTox-ONE TM Assay. Cells were digested in the serum-free medium ASF Medium 104 for 4 days by adding 5 munits/well of Chase ABC twice at 0 and 2 days. (F) Cells digested with or without Chase ABC were stained by anti-CS antibody (clone 2B6), which detects the terminal unsaturated disaccharide of CS chains generated by Chase ABC. (G) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without GM6001 was measured. Statistical significance was determined using Student’s t -test. Statistical analyses were performed using KaleidaGraph version 4.5.1.
    Human Breast Cancer Cell Line Bt 549, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human breast cancer cell line bt549 cells
    C4ST-1 deficiency suppresses the proliferation of <t>BT-549</t> cells. (A) Growth curves of parental BT-549 and C4ST-1 KO cells. (B) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) was measured by CytoTox-ONE TM Assay. (C) Proliferation of BT-549 and C4ST-1 KO cells was examined by colony formation assay. Both the cell types were seeded at a concentration of 50 cells/well in 6-well plate and cultured for 9 days. The colonies were stained with crystal violet, and observed under a light microscope (Left). The number of colonies of BT-549 ( n = 3) and C4ST-1 KO cells ( n = 3) was compared (Right). (D) The level of cyclin D1 in BT-549 and C4ST-1 KO cells was measured by real-time PCR ( n = 3 each). (E) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without Chase ABC was examined by CytoTox-ONE TM Assay. Cells were digested in the serum-free medium ASF Medium 104 for 4 days by adding 5 munits/well of Chase ABC twice at 0 and 2 days. (F) Cells digested with or without Chase ABC were stained by anti-CS antibody (clone 2B6), which detects the terminal unsaturated disaccharide of CS chains generated by Chase ABC. (G) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without GM6001 was measured. Statistical significance was determined using Student’s t -test. Statistical analyses were performed using KaleidaGraph version 4.5.1.
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    qRT-PCR analysis for FILIP1L conducted on cDNA from BT-549 breast ( A ), HT-29 colon ( B ), H1299 lung ( C ) and MIA PaCa-2 pancreatic ( D ) cancer cell lines treated with either 5-aza-2’-deoxycytidine (DAC) or Trichostatin A (TSA). Values 0.1, 1 or 10 in the x axis indicate the concentration of DAC and TSA in μM. The y axis represents fold change of each reagent-treated cell type over DMSO-treated control cells, where each value was standardized with the housekeeping gene hRPL7 . Error bars indicate SEM ( n = 3). The result is an average of two independent experiments. P values are derived from comparison between DMSO-treated control and either DAC- or TSA-treated experiments. NS indicates not significant.

    Journal: PLoS ONE

    Article Title: Down-Regulation of Fil amin A i nteracting p rotein 1 - l ike Is Associated with Promoter Methylation and an Invasive Phenotype in Breast, Colon, Lung and Pancreatic Cancers

    doi: 10.1371/journal.pone.0082620

    Figure Lengend Snippet: qRT-PCR analysis for FILIP1L conducted on cDNA from BT-549 breast ( A ), HT-29 colon ( B ), H1299 lung ( C ) and MIA PaCa-2 pancreatic ( D ) cancer cell lines treated with either 5-aza-2’-deoxycytidine (DAC) or Trichostatin A (TSA). Values 0.1, 1 or 10 in the x axis indicate the concentration of DAC and TSA in μM. The y axis represents fold change of each reagent-treated cell type over DMSO-treated control cells, where each value was standardized with the housekeeping gene hRPL7 . Error bars indicate SEM ( n = 3). The result is an average of two independent experiments. P values are derived from comparison between DMSO-treated control and either DAC- or TSA-treated experiments. NS indicates not significant.

    Article Snippet: Human pancreatic cancer cell lines MIA PaCa-2, PANC-1, Hs 766T, HPAC, HPAF-II, SU.86.86, Panc 02.03 and Capan-1 and human breast cancer cell lines BT-549, Hs 578T, MDA-MB-468, BT-474 and ZR-75-1 were purchased from ATCC.

    Techniques: Quantitative RT-PCR, Concentration Assay, Derivative Assay

    C4ST-1 deficiency suppresses the proliferation of BT-549 cells. (A) Growth curves of parental BT-549 and C4ST-1 KO cells. (B) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) was measured by CytoTox-ONE TM Assay. (C) Proliferation of BT-549 and C4ST-1 KO cells was examined by colony formation assay. Both the cell types were seeded at a concentration of 50 cells/well in 6-well plate and cultured for 9 days. The colonies were stained with crystal violet, and observed under a light microscope (Left). The number of colonies of BT-549 ( n = 3) and C4ST-1 KO cells ( n = 3) was compared (Right). (D) The level of cyclin D1 in BT-549 and C4ST-1 KO cells was measured by real-time PCR ( n = 3 each). (E) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without Chase ABC was examined by CytoTox-ONE TM Assay. Cells were digested in the serum-free medium ASF Medium 104 for 4 days by adding 5 munits/well of Chase ABC twice at 0 and 2 days. (F) Cells digested with or without Chase ABC were stained by anti-CS antibody (clone 2B6), which detects the terminal unsaturated disaccharide of CS chains generated by Chase ABC. (G) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without GM6001 was measured. Statistical significance was determined using Student’s t -test. Statistical analyses were performed using KaleidaGraph version 4.5.1.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation

    doi: 10.3389/fcell.2021.659428

    Figure Lengend Snippet: C4ST-1 deficiency suppresses the proliferation of BT-549 cells. (A) Growth curves of parental BT-549 and C4ST-1 KO cells. (B) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) was measured by CytoTox-ONE TM Assay. (C) Proliferation of BT-549 and C4ST-1 KO cells was examined by colony formation assay. Both the cell types were seeded at a concentration of 50 cells/well in 6-well plate and cultured for 9 days. The colonies were stained with crystal violet, and observed under a light microscope (Left). The number of colonies of BT-549 ( n = 3) and C4ST-1 KO cells ( n = 3) was compared (Right). (D) The level of cyclin D1 in BT-549 and C4ST-1 KO cells was measured by real-time PCR ( n = 3 each). (E) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without Chase ABC was examined by CytoTox-ONE TM Assay. Cells were digested in the serum-free medium ASF Medium 104 for 4 days by adding 5 munits/well of Chase ABC twice at 0 and 2 days. (F) Cells digested with or without Chase ABC were stained by anti-CS antibody (clone 2B6), which detects the terminal unsaturated disaccharide of CS chains generated by Chase ABC. (G) Proliferation of BT-549 ( n = 4) and C4ST-1 KO cells ( n = 4) treated with or without GM6001 was measured. Statistical significance was determined using Student’s t -test. Statistical analyses were performed using KaleidaGraph version 4.5.1.

    Article Snippet: The human breast cancer cell line BT-549 (ATCC ® HTB-122 TM ), ER-, and ERBB2-negative (triple-negative and basal B subtype) breast cancer cell lines were obtained from American Type Culture Collection (ATCC) ( ; ).

    Techniques: Colony Assay, Concentration Assay, Cell Culture, Staining, Light Microscopy, Real-time Polymerase Chain Reaction, Generated

    Inhibition of the cleavage of SDC1 by loss of C4ST-1 expression. (A) Schematic representation of the cleavage sites of MMPs, HS/CS attachment sites on SDC1, and the epitope of the anti-SDC1 antibody used in this study. CS chains are indicated by dashed lines because Ser 206 and Ser 216 are not modified with CS chains in BT-549 cells. In addition, CS chains are attached to Ser 37 , Ser 45 , or Ser 47 , because a SDC1 core protein is modified with CS chains according to our previous study . Anti-SDC1 antibody used in this study recognizes the SDC1 fragment (approx. 32.5 kDa) after cleavage by MMPs. (B) BT-549 and C4ST-1 KO cells were incubated in the presence (+) or absence (−) of GM6001. Cell lysates and conditioned medium prepared from each cell were treated with (+) or without (−) Chase ABC, HSase, and Hepase, and then subject to immunoblotting using the anti-SDC1 antibody. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. (C) The surface expression of SDC1 in BT-549 and BT-549 treated with GM6001, and C4ST-1KO cells were examined by flow cytometry. (D) The expression level of MMP2, MMP9, and MMP14 in BT-549 and C4ST-1KO cells was examined by immunoblotting. (E) The levels of MMP2, MMP7, MMP9, and MMP14 in BT-549 cells transfected either with si-Control or si-MMP2, 7, 9, or 14 were measured by real-time PCR. (F) The effect of knockdown of MMP2, 7, 9, or 14 on the cleavage of SDC1 was examined. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. At the right side, the ratio of cleaved SDC1 to full-length SDC1 is shown as fold change relative to that of siControl. (G) The effect of knockdown of MMP2, MMP7, MMP9, and MMP14 on the proliferation of BT-549 cells ( n = 4, each) was examined by CytoTox-ONE TM Assay. (H) The effect of knockdown of β-catenin on the proliferation of BT-549 cells ( n = 6) was investigated. Statistical significance was determined using Student’s t -test.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation

    doi: 10.3389/fcell.2021.659428

    Figure Lengend Snippet: Inhibition of the cleavage of SDC1 by loss of C4ST-1 expression. (A) Schematic representation of the cleavage sites of MMPs, HS/CS attachment sites on SDC1, and the epitope of the anti-SDC1 antibody used in this study. CS chains are indicated by dashed lines because Ser 206 and Ser 216 are not modified with CS chains in BT-549 cells. In addition, CS chains are attached to Ser 37 , Ser 45 , or Ser 47 , because a SDC1 core protein is modified with CS chains according to our previous study . Anti-SDC1 antibody used in this study recognizes the SDC1 fragment (approx. 32.5 kDa) after cleavage by MMPs. (B) BT-549 and C4ST-1 KO cells were incubated in the presence (+) or absence (−) of GM6001. Cell lysates and conditioned medium prepared from each cell were treated with (+) or without (−) Chase ABC, HSase, and Hepase, and then subject to immunoblotting using the anti-SDC1 antibody. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. (C) The surface expression of SDC1 in BT-549 and BT-549 treated with GM6001, and C4ST-1KO cells were examined by flow cytometry. (D) The expression level of MMP2, MMP9, and MMP14 in BT-549 and C4ST-1KO cells was examined by immunoblotting. (E) The levels of MMP2, MMP7, MMP9, and MMP14 in BT-549 cells transfected either with si-Control or si-MMP2, 7, 9, or 14 were measured by real-time PCR. (F) The effect of knockdown of MMP2, 7, 9, or 14 on the cleavage of SDC1 was examined. Arrow and open triangle indicate the core protein of full-length SDC1 and the SDC1 fragment after cleavage by MMPs, respectively. At the right side, the ratio of cleaved SDC1 to full-length SDC1 is shown as fold change relative to that of siControl. (G) The effect of knockdown of MMP2, MMP7, MMP9, and MMP14 on the proliferation of BT-549 cells ( n = 4, each) was examined by CytoTox-ONE TM Assay. (H) The effect of knockdown of β-catenin on the proliferation of BT-549 cells ( n = 6) was investigated. Statistical significance was determined using Student’s t -test.

    Article Snippet: The human breast cancer cell line BT-549 (ATCC ® HTB-122 TM ), ER-, and ERBB2-negative (triple-negative and basal B subtype) breast cancer cell lines were obtained from American Type Culture Collection (ATCC) ( ; ).

    Techniques: Inhibition, Expressing, Modification, Incubation, Western Blot, Flow Cytometry, Transfection, Real-time Polymerase Chain Reaction

    Suppression of SUMOylation of AKT1 by the loss of C4ST-1 expression. (A) Some of the signaling pathways involved in cancer proliferation examined in this study are shown. (B) Phosphorylation of PI3K, AKT1, S6K, ERK1/2, and STAT3 in BT-549 and C4ST-1 KO cells was examined by immunoblotting using phospho-specific antibodies and total antibodies. (C) Phosphorylation of PI3K ( n = 3), AKT1 ( n = 5), S6K ( n = 4), ERK1/2 ( n = 4), and STAT3 ( n = 4) in BT-549 and C4ST-1 KO cells was quantified by calculating the ratios of phosphorylated to total protein. (D) The effect of tannic acid and AKT inhibitor (GSK690693) on the proliferation of BT-549 and C4ST-1KO cells. (E) SUMOylation of AKT1 in BT-549 and C4ST-1 KO cells were examined. Both the cells were treated with (+) or without (−) GM6001, and lysed in the absence (−) or presence (+) of N -ethylmaleimide (NEM), which inhibits SUMO proteases. Immunoprecipitated phospho-AKT1 proteins were subject to immunoblotting using anti-pAkt1 and anti-SUMO1 antibodies.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation

    doi: 10.3389/fcell.2021.659428

    Figure Lengend Snippet: Suppression of SUMOylation of AKT1 by the loss of C4ST-1 expression. (A) Some of the signaling pathways involved in cancer proliferation examined in this study are shown. (B) Phosphorylation of PI3K, AKT1, S6K, ERK1/2, and STAT3 in BT-549 and C4ST-1 KO cells was examined by immunoblotting using phospho-specific antibodies and total antibodies. (C) Phosphorylation of PI3K ( n = 3), AKT1 ( n = 5), S6K ( n = 4), ERK1/2 ( n = 4), and STAT3 ( n = 4) in BT-549 and C4ST-1 KO cells was quantified by calculating the ratios of phosphorylated to total protein. (D) The effect of tannic acid and AKT inhibitor (GSK690693) on the proliferation of BT-549 and C4ST-1KO cells. (E) SUMOylation of AKT1 in BT-549 and C4ST-1 KO cells were examined. Both the cells were treated with (+) or without (−) GM6001, and lysed in the absence (−) or presence (+) of N -ethylmaleimide (NEM), which inhibits SUMO proteases. Immunoprecipitated phospho-AKT1 proteins were subject to immunoblotting using anti-pAkt1 and anti-SUMO1 antibodies.

    Article Snippet: The human breast cancer cell line BT-549 (ATCC ® HTB-122 TM ), ER-, and ERBB2-negative (triple-negative and basal B subtype) breast cancer cell lines were obtained from American Type Culture Collection (ATCC) ( ; ).

    Techniques: Expressing, Western Blot, Immunoprecipitation

    Cellular localization of SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG and the effect of SDC1 fragments on cell proliferation after cleavage of MMPs. (A) SDC1(FL)-3xFLAG, SDC1(NTF)-3xFLAG, and SDC1(CTF)-3xFLAG were schematically illustrated. Recognition sites of anti-SDC1 antibodies used in this study are shown. (B) The Expression of SDC1(NTF)-3xFLAG was confirmed by immunoblotting. Conditioned medium and cell lysate was digested with (+) or without (−) GAGase (the mixture of Chase ABC, HSase, and Hepase). SDC1(NTF)-3xFLAG was detected in medium digested with GAGase. (C) The expression of SDC1(FL)-3xFLAG or SDC1(CTF)-3xFLAG in stable clones of BT-549 cells overexpressing SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was confirmed by immunoblotting. Each cell lysate was digested with (+) or without (−) GAGase (the mixture of chondroitinase ABC, heparitinase, and heparinase). SDC1(FL)-3xFLAG modified with GAG chains is represented by “SDC1(FL) + GAG.” BT-549 cells stably expressing the empty vector p3xFLAG-CMV14 is represented as “empty.” (D) Expression pattern of exogenously expressed SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was compared with that of endogenous SDC1 by immunofluorescence method using anti-SDC1 antibodies (HPA00618 and D4Y7H) and anti-FLAG antibody. (E) Proliferation of BT-549 cells overexpressing the empty vector ( n = 4) and SDC1(NTF)-3xFLAG ( n = 3), or proliferation of BT-549 cells overexpressing the empty vector ( n = 5), SDC1(FL)-3xFLAG ( n = 5), and SDC1(CTF)-3xFLAG ( n = 5) was measured.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation

    doi: 10.3389/fcell.2021.659428

    Figure Lengend Snippet: Cellular localization of SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG and the effect of SDC1 fragments on cell proliferation after cleavage of MMPs. (A) SDC1(FL)-3xFLAG, SDC1(NTF)-3xFLAG, and SDC1(CTF)-3xFLAG were schematically illustrated. Recognition sites of anti-SDC1 antibodies used in this study are shown. (B) The Expression of SDC1(NTF)-3xFLAG was confirmed by immunoblotting. Conditioned medium and cell lysate was digested with (+) or without (−) GAGase (the mixture of Chase ABC, HSase, and Hepase). SDC1(NTF)-3xFLAG was detected in medium digested with GAGase. (C) The expression of SDC1(FL)-3xFLAG or SDC1(CTF)-3xFLAG in stable clones of BT-549 cells overexpressing SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was confirmed by immunoblotting. Each cell lysate was digested with (+) or without (−) GAGase (the mixture of chondroitinase ABC, heparitinase, and heparinase). SDC1(FL)-3xFLAG modified with GAG chains is represented by “SDC1(FL) + GAG.” BT-549 cells stably expressing the empty vector p3xFLAG-CMV14 is represented as “empty.” (D) Expression pattern of exogenously expressed SDC1(FL)-3xFLAG and SDC1(CTF)-3xFLAG was compared with that of endogenous SDC1 by immunofluorescence method using anti-SDC1 antibodies (HPA00618 and D4Y7H) and anti-FLAG antibody. (E) Proliferation of BT-549 cells overexpressing the empty vector ( n = 4) and SDC1(NTF)-3xFLAG ( n = 3), or proliferation of BT-549 cells overexpressing the empty vector ( n = 5), SDC1(FL)-3xFLAG ( n = 5), and SDC1(CTF)-3xFLAG ( n = 5) was measured.

    Article Snippet: The human breast cancer cell line BT-549 (ATCC ® HTB-122 TM ), ER-, and ERBB2-negative (triple-negative and basal B subtype) breast cancer cell lines were obtained from American Type Culture Collection (ATCC) ( ; ).

    Techniques: Expressing, Western Blot, Clone Assay, Modification, Stable Transfection, Plasmid Preparation, Immunofluorescence

    Enhancement of cell proliferation through the SUMOylation of AKT1 upregulated by the expression of the C-terminal fragment of SDC1. (A) The effect of GM6001 on the proliferation of BT-549 cells overexpressing the empty vector ( n = 5), SDC1(FL)-3xFLAG ( n = 5), and SDC1(CTF)-3xFLAG ( n = 5) was examined. (B) The effect of tannic acid on the proliferation of BT-549 cells overexpressing the empty vector ( n = 4), SDC1(FL)-3xFLAG ( n = 4), and SDC1(CTF)-3xFLAG ( n = 4) was examined. (C) SUMOylated proteins in BT-549 cells overexpressing the empty vector and SDC1(CTF)-3xFLAG were captured using the SUMO-QAPTURE-T kit, and subject to immunoblotting using the anti-Akt1 and anti-SUMO1 antibodies.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation

    doi: 10.3389/fcell.2021.659428

    Figure Lengend Snippet: Enhancement of cell proliferation through the SUMOylation of AKT1 upregulated by the expression of the C-terminal fragment of SDC1. (A) The effect of GM6001 on the proliferation of BT-549 cells overexpressing the empty vector ( n = 5), SDC1(FL)-3xFLAG ( n = 5), and SDC1(CTF)-3xFLAG ( n = 5) was examined. (B) The effect of tannic acid on the proliferation of BT-549 cells overexpressing the empty vector ( n = 4), SDC1(FL)-3xFLAG ( n = 4), and SDC1(CTF)-3xFLAG ( n = 4) was examined. (C) SUMOylated proteins in BT-549 cells overexpressing the empty vector and SDC1(CTF)-3xFLAG were captured using the SUMO-QAPTURE-T kit, and subject to immunoblotting using the anti-Akt1 and anti-SUMO1 antibodies.

    Article Snippet: The human breast cancer cell line BT-549 (ATCC ® HTB-122 TM ), ER-, and ERBB2-negative (triple-negative and basal B subtype) breast cancer cell lines were obtained from American Type Culture Collection (ATCC) ( ; ).

    Techniques: Expressing, Plasmid Preparation, Western Blot

    C4ST-1 controls BT-549 cell proliferation by regulating the cleavage of SDC1 by MMPs. SDC1 expressed at the cell surface is cleaved by MMPs, and the N-terminal and the C-terminal fragments of SDC1 are generated. Both fragments, SDC1(NTF) and SDC1(CTF), have a positive effect on cell proliferation, and cell proliferation is more affected by SDC1(CTF) than by SDC1(NTF). SDC1(CTF) is involved in SUMOylation of AKT1, although the mechanism underlying SUMOylation by SDC1(CTF) remains unclear. SUMOylation of AKT1 enhances proliferation through S6 kinase signaling pathway. In C4ST-1KO cells, the cleavage of SDC1 by MMPs is suppressed because of reduced expression of MMPs. SUMOylation of AKT1 is inhibited because of decreased SDC1(CTF), and proliferation is slowed down.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation

    doi: 10.3389/fcell.2021.659428

    Figure Lengend Snippet: C4ST-1 controls BT-549 cell proliferation by regulating the cleavage of SDC1 by MMPs. SDC1 expressed at the cell surface is cleaved by MMPs, and the N-terminal and the C-terminal fragments of SDC1 are generated. Both fragments, SDC1(NTF) and SDC1(CTF), have a positive effect on cell proliferation, and cell proliferation is more affected by SDC1(CTF) than by SDC1(NTF). SDC1(CTF) is involved in SUMOylation of AKT1, although the mechanism underlying SUMOylation by SDC1(CTF) remains unclear. SUMOylation of AKT1 enhances proliferation through S6 kinase signaling pathway. In C4ST-1KO cells, the cleavage of SDC1 by MMPs is suppressed because of reduced expression of MMPs. SUMOylation of AKT1 is inhibited because of decreased SDC1(CTF), and proliferation is slowed down.

    Article Snippet: The human breast cancer cell line BT-549 (ATCC ® HTB-122 TM ), ER-, and ERBB2-negative (triple-negative and basal B subtype) breast cancer cell lines were obtained from American Type Culture Collection (ATCC) ( ; ).

    Techniques: Generated, Expressing