breast cancer cell line mda mb  (ATCC)


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    ATCC breast cancer cell line mda mb
    Breast Cancer Cell Line Mda Mb, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    breast cancer cell line mda mb 435  (ATCC)


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    ATCC breast cancer cell line mda mb 435
    Breast Cancer Cell Line Mda Mb 435, 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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    breast cancer cell line mda mb 435  (ATCC)


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    ATCC breast cancer cell line mda mb 435
    Breast Cancer Cell Line Mda Mb 435, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human breast cancer cells mda mb 435 melanoma cell line meoh methanol mgc  (ATCC)


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    ATCC human breast cancer cells mda mb 435 melanoma cell line meoh methanol mgc
    Human Breast Cancer Cells Mda Mb 435 Melanoma Cell Line Meoh Methanol Mgc, 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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    breast cancer cell line mda mb  (ATCC)


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    ATCC breast cancer cell line mda mb
    Breast Cancer Cell Line Mda Mb, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human breast cancer cell lines htb129  (ATCC)


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    ATCC human breast cancer cell lines htb129
    <t>HTB129-ctrl</t> and HTB129-miR203 cells were subjected to A) qRT-PCR analyses of SNAI1 mRNA expression in HTB129-ctrl and HTB129-miR203 cells, B) fluorescent staining with DAPI (blue) and phalloidin (red) (scale bar, 20 µm), C) migration and D) invasion assays. E) Predicted miR-203 target sites within SNAI1 3′UTR. F, G) Relative luciferase activity of SNAI1-3′UTR wild type (wt) or mutant (mut) in MDA231 cells transfected with control (F, G), miR-203 (F) or miR-200a/c (G) precursors. Co-transfection with GAPDH 3′UTR vector served as additional negative control (*, p<0.05; **, p<0.01).
    Human Breast Cancer Cell Lines Htb129, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A Novel Network Integrating a miRNA-203/SNAI1 Feedback Loop which Regulates Epithelial to Mesenchymal Transition"

    Article Title: A Novel Network Integrating a miRNA-203/SNAI1 Feedback Loop which Regulates Epithelial to Mesenchymal Transition

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035440

    HTB129-ctrl and HTB129-miR203 cells were subjected to A) qRT-PCR analyses of SNAI1 mRNA expression in HTB129-ctrl and HTB129-miR203 cells, B) fluorescent staining with DAPI (blue) and phalloidin (red) (scale bar, 20 µm), C) migration and D) invasion assays. E) Predicted miR-203 target sites within SNAI1 3′UTR. F, G) Relative luciferase activity of SNAI1-3′UTR wild type (wt) or mutant (mut) in MDA231 cells transfected with control (F, G), miR-203 (F) or miR-200a/c (G) precursors. Co-transfection with GAPDH 3′UTR vector served as additional negative control (*, p<0.05; **, p<0.01).
    Figure Legend Snippet: HTB129-ctrl and HTB129-miR203 cells were subjected to A) qRT-PCR analyses of SNAI1 mRNA expression in HTB129-ctrl and HTB129-miR203 cells, B) fluorescent staining with DAPI (blue) and phalloidin (red) (scale bar, 20 µm), C) migration and D) invasion assays. E) Predicted miR-203 target sites within SNAI1 3′UTR. F, G) Relative luciferase activity of SNAI1-3′UTR wild type (wt) or mutant (mut) in MDA231 cells transfected with control (F, G), miR-203 (F) or miR-200a/c (G) precursors. Co-transfection with GAPDH 3′UTR vector served as additional negative control (*, p<0.05; **, p<0.01).

    Techniques Used: Quantitative RT-PCR, Expressing, Staining, Migration, Luciferase, Activity Assay, Mutagenesis, Transfection, Cotransfection, Plasmid Preparation, Negative Control

    mda mb 435s breast cancer cell lines  (ATCC)


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    ATCC mda mb 435s breast cancer cell lines
    A . Experion Bioanalyser gel image for RNA isolated following immunoprecipitation with YB-1 antibody or IgG isotype control antibody (labelled YB-1 IP and IgG IP respectively) from <t>MDA-MB-435S</t> cells. Ribosomal RNAs (18S and 28S) are shown by arrows. The ladder shows the size of the RNAs in nucleotides. Small RNAs are highlighted by the labelled bracket. B . RT-qPCR detection of mRNAs bound following immunoprecipitation with the YB-1 antibody.
    Mda Mb 435s Breast Cancer Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Links between the Oncoprotein YB-1 and Small Non-Coding RNAs in Breast Cancer"

    Article Title: Links between the Oncoprotein YB-1 and Small Non-Coding RNAs in Breast Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080171

    A . Experion Bioanalyser gel image for RNA isolated following immunoprecipitation with YB-1 antibody or IgG isotype control antibody (labelled YB-1 IP and IgG IP respectively) from MDA-MB-435S cells. Ribosomal RNAs (18S and 28S) are shown by arrows. The ladder shows the size of the RNAs in nucleotides. Small RNAs are highlighted by the labelled bracket. B . RT-qPCR detection of mRNAs bound following immunoprecipitation with the YB-1 antibody.
    Figure Legend Snippet: A . Experion Bioanalyser gel image for RNA isolated following immunoprecipitation with YB-1 antibody or IgG isotype control antibody (labelled YB-1 IP and IgG IP respectively) from MDA-MB-435S cells. Ribosomal RNAs (18S and 28S) are shown by arrows. The ladder shows the size of the RNAs in nucleotides. Small RNAs are highlighted by the labelled bracket. B . RT-qPCR detection of mRNAs bound following immunoprecipitation with the YB-1 antibody.

    Techniques Used: Isolation, Immunoprecipitation, Quantitative RT-PCR

    sncRNAs that are bound by YB-1 protein by immunoprecipitation in MCF7 and  MDA-MB-435S  cells based on enrichment in abundance in IP over input.
    Figure Legend Snippet: sncRNAs that are bound by YB-1 protein by immunoprecipitation in MCF7 and MDA-MB-435S cells based on enrichment in abundance in IP over input.

    Techniques Used: Immunoprecipitation

    A. MCF7 cells B. MDA-MB-435S cells Note: miR-886 abundance in MCF7 cells was undetectable.
    Figure Legend Snippet: A. MCF7 cells B. MDA-MB-435S cells Note: miR-886 abundance in MCF7 cells was undetectable.

    Techniques Used:

    triple negative breast cancer cell line mda mb 435  (ATCC)


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    ATCC triple negative breast cancer cell line mda mb 435
    MiR-27a is significantly increased and CDC27 expression is significantly decreased in <t>TNBC</t> cells. ( A ) qRT-PCR analysis of miR-27a expression in <t>MDA-MB-435,</t> MDA-MB-231, and MCF10A cell lines. ( B ) qRT-PCR analysis of CDC27 mRNA expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. ( C ) Western blot analysis of CDC27 expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. Data are shown as mean±S.D by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.
    Triple Negative Breast Cancer Cell Line Mda Mb 435, 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 "MiR-27a Modulates Radiosensitivity of Triple-Negative Breast Cancer (TNBC) Cells by Targeting CDC27"

    Article Title: MiR-27a Modulates Radiosensitivity of Triple-Negative Breast Cancer (TNBC) Cells by Targeting CDC27

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.893974

    MiR-27a is significantly increased and CDC27 expression is significantly decreased in TNBC cells. ( A ) qRT-PCR analysis of miR-27a expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. ( B ) qRT-PCR analysis of CDC27 mRNA expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. ( C ) Western blot analysis of CDC27 expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. Data are shown as mean±S.D by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.
    Figure Legend Snippet: MiR-27a is significantly increased and CDC27 expression is significantly decreased in TNBC cells. ( A ) qRT-PCR analysis of miR-27a expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. ( B ) qRT-PCR analysis of CDC27 mRNA expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. ( C ) Western blot analysis of CDC27 expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. Data are shown as mean±S.D by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

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

    MiR-27a is involved in radiosensitivity of TNBC cells. ( A–D ) qRT-PCR analysis of miR-27a expression in MDA-MB-435 ( A, C ) MDA-MB-231 ( B, D ) cells after transfection of 200nM antagomiR-27a ( A, B ) 75nM miR-27a mimics (C, D). ( D–G ) CCK-8 assay of cell proliferation of MDA-MB-435 ( D, F ) MDA-MB-231 ( E, G ) cells transfected with 200 nM antagomiR-27a with/without treatment of IR (8 Gy) (E, F) or transfected with 75 nM miR-27a mimics with/without treatment of IR (8 Gy) ( G, H ). ( I, J ) MDA-MB-435 cells with miR-27a knockdown or overexpression were stained with caspase-3 staining kit 24 h after IR (8 Gy) treatment. Then, flow cytometry analysis was performed to measure the proportion of cells with active caspase-3 staining. Representative flow cytometry images of the cells with active caspase-3 ( I ). Quantification of apoptotic cells with active caspase-3 signal in ( J ). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.
    Figure Legend Snippet: MiR-27a is involved in radiosensitivity of TNBC cells. ( A–D ) qRT-PCR analysis of miR-27a expression in MDA-MB-435 ( A, C ) MDA-MB-231 ( B, D ) cells after transfection of 200nM antagomiR-27a ( A, B ) 75nM miR-27a mimics (C, D). ( D–G ) CCK-8 assay of cell proliferation of MDA-MB-435 ( D, F ) MDA-MB-231 ( E, G ) cells transfected with 200 nM antagomiR-27a with/without treatment of IR (8 Gy) (E, F) or transfected with 75 nM miR-27a mimics with/without treatment of IR (8 Gy) ( G, H ). ( I, J ) MDA-MB-435 cells with miR-27a knockdown or overexpression were stained with caspase-3 staining kit 24 h after IR (8 Gy) treatment. Then, flow cytometry analysis was performed to measure the proportion of cells with active caspase-3 staining. Representative flow cytometry images of the cells with active caspase-3 ( I ). Quantification of apoptotic cells with active caspase-3 signal in ( J ). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Over Expression, Staining, Flow Cytometry

    MiR-27a directly targets CDC27 and regulates its expression in TNBC cells. ( A ) The putative binding site between miR-27a and CDC27 and the designed mutant sequence without the pairing. ( B, C ) The relative firefly luciferase activity in HEK-293T ( B ) and MDA-MB-435 ( C ) cells co-transfected with 150 ng reporter plasmids and 50 nM miR-27a mimics. Both firefly and Renilla luciferase activities were measured 24 h after transfection and the firefly luciferase activity was normalized to the Renilla luciferase activity. ( D, E ) Western blot analysis of CDC27 protein expression in MDA-MB-435 transfected with miR-27a mimics or siCDC-27 ( D ) or transfected with antagomiR-27a or infected with CDC-27 lentiviral particles ( E ). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.
    Figure Legend Snippet: MiR-27a directly targets CDC27 and regulates its expression in TNBC cells. ( A ) The putative binding site between miR-27a and CDC27 and the designed mutant sequence without the pairing. ( B, C ) The relative firefly luciferase activity in HEK-293T ( B ) and MDA-MB-435 ( C ) cells co-transfected with 150 ng reporter plasmids and 50 nM miR-27a mimics. Both firefly and Renilla luciferase activities were measured 24 h after transfection and the firefly luciferase activity was normalized to the Renilla luciferase activity. ( D, E ) Western blot analysis of CDC27 protein expression in MDA-MB-435 transfected with miR-27a mimics or siCDC-27 ( D ) or transfected with antagomiR-27a or infected with CDC-27 lentiviral particles ( E ). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

    Techniques Used: Expressing, Binding Assay, Mutagenesis, Sequencing, Luciferase, Activity Assay, Transfection, Western Blot, Infection

    MiR-27a modulates radiosensitivity of TNBC cells through targeting CDC27. ( A–D ) CCK-8 assay of cell proliferation in MDA-MB-435 ( A, C ) MDA-MB-231 ( B, D ) cells after infection with CDC27 lentiviral particle with/without treatment of IR (8 Gy) ( A, B ) or transfected with 50 nM siCDC27 with/without treatment of IR (8 Gy) ( C, D ). ( E, F ) The effect of co-transfection of miR-27a and CDC27 on cell proliferation in MDA-MB-435 ( E ) MDA-MB-231 ( F ) cells with/without treatment of IR (8 Gy). ( G, H ) The effect of co-transfection of antagomiR-27a and siCDC27 on cell proliferation in MDA-MB-435 ( G ) MDA-MB-231 ( H ) cells with/without treatment of IR (8 Gy). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.
    Figure Legend Snippet: MiR-27a modulates radiosensitivity of TNBC cells through targeting CDC27. ( A–D ) CCK-8 assay of cell proliferation in MDA-MB-435 ( A, C ) MDA-MB-231 ( B, D ) cells after infection with CDC27 lentiviral particle with/without treatment of IR (8 Gy) ( A, B ) or transfected with 50 nM siCDC27 with/without treatment of IR (8 Gy) ( C, D ). ( E, F ) The effect of co-transfection of miR-27a and CDC27 on cell proliferation in MDA-MB-435 ( E ) MDA-MB-231 ( F ) cells with/without treatment of IR (8 Gy). ( G, H ) The effect of co-transfection of antagomiR-27a and siCDC27 on cell proliferation in MDA-MB-435 ( G ) MDA-MB-231 ( H ) cells with/without treatment of IR (8 Gy). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

    Techniques Used: CCK-8 Assay, Infection, Transfection, Cotransfection

    human breast cancer cell lines mda mb 435  (ATCC)


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    ATCC human breast cancer cell lines mda mb 435
    Expression of TFPI-2 in human breast cancer cells with different metastasis potential . (A). Western blot analysis of TFPI-2 protein expression. The HUVEC cell line was used as positive control and the protein levels of GAPDH were determined as control. (B). Quantitative real-time PCR analysis of TFPI-2 mRNA levels. All expression levels of TFPI-2 in breast cancer cell lines were normalized to the level of its expression in HUVEC. Lane 1: <t>MDA-MB-435.</t> Lane 2: T47D. Lane 3: MCF-7. Lane 4: HUVEC.
    Human Breast Cancer Cell Lines Mda Mb 435, 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 "Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells"

    Article Title: Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-8-110

    Expression of TFPI-2 in human breast cancer cells with different metastasis potential . (A). Western blot analysis of TFPI-2 protein expression. The HUVEC cell line was used as positive control and the protein levels of GAPDH were determined as control. (B). Quantitative real-time PCR analysis of TFPI-2 mRNA levels. All expression levels of TFPI-2 in breast cancer cell lines were normalized to the level of its expression in HUVEC. Lane 1: MDA-MB-435. Lane 2: T47D. Lane 3: MCF-7. Lane 4: HUVEC.
    Figure Legend Snippet: Expression of TFPI-2 in human breast cancer cells with different metastasis potential . (A). Western blot analysis of TFPI-2 protein expression. The HUVEC cell line was used as positive control and the protein levels of GAPDH were determined as control. (B). Quantitative real-time PCR analysis of TFPI-2 mRNA levels. All expression levels of TFPI-2 in breast cancer cell lines were normalized to the level of its expression in HUVEC. Lane 1: MDA-MB-435. Lane 2: T47D. Lane 3: MCF-7. Lane 4: HUVEC.

    Techniques Used: Expressing, Western Blot, Positive Control, Real-time Polymerase Chain Reaction

    Analysis of TFPI-2 promoter . (A) Breast cancer cell lines were transfected with promoter-luciferase plasmid cloned from different cell lines, bearing respective genetic mutation. The promoter from different cell line has nearly the same luciferase activity in both MDA-MB-435 and MCF-7 cell lines. Results are shown as mean ± SD. The apparent lower level of promoter activity in MDA-MB-435 cell was not statistically significant (p = 0.342>0.05). (B) Luciferase reporter gene constructs with 5'ends between nucleotides -1436 and -144 and a common 3'end at +75 were transiently transfected into breast cancer cells. The minimal construct P-144 has the same luciferase activity as P-1436. (C) Linker-scan mutation analysis of TFPI-2 promoter -144 to +1 region. The fragment from -144 to the transcriptional start point (+1) of TFPI-2 promoter were systematically replaced with an BamH I & Xba I polylinker (GGATCCTCTAGA), the resulting sequence were analyzed for not introducing new transcription factor binding site by MatInspector [21]. SM1 indicate the promoter sequence from -12 to +1, and in turn every 12 bases from -144 to -12 was indicated by SM12 to SM2. Then the luciferase promoter reporter plasmids were transfected into MDA-MB-435 cell line and the reported luciferase activity was measured. Results are shown as mean ± SD relative to the wild type (*p < 0.05; **p < 0.001).
    Figure Legend Snippet: Analysis of TFPI-2 promoter . (A) Breast cancer cell lines were transfected with promoter-luciferase plasmid cloned from different cell lines, bearing respective genetic mutation. The promoter from different cell line has nearly the same luciferase activity in both MDA-MB-435 and MCF-7 cell lines. Results are shown as mean ± SD. The apparent lower level of promoter activity in MDA-MB-435 cell was not statistically significant (p = 0.342>0.05). (B) Luciferase reporter gene constructs with 5'ends between nucleotides -1436 and -144 and a common 3'end at +75 were transiently transfected into breast cancer cells. The minimal construct P-144 has the same luciferase activity as P-1436. (C) Linker-scan mutation analysis of TFPI-2 promoter -144 to +1 region. The fragment from -144 to the transcriptional start point (+1) of TFPI-2 promoter were systematically replaced with an BamH I & Xba I polylinker (GGATCCTCTAGA), the resulting sequence were analyzed for not introducing new transcription factor binding site by MatInspector [21]. SM1 indicate the promoter sequence from -12 to +1, and in turn every 12 bases from -144 to -12 was indicated by SM12 to SM2. Then the luciferase promoter reporter plasmids were transfected into MDA-MB-435 cell line and the reported luciferase activity was measured. Results are shown as mean ± SD relative to the wild type (*p < 0.05; **p < 0.001).

    Techniques Used: Transfection, Luciferase, Plasmid Preparation, Clone Assay, Mutagenesis, Activity Assay, Construct, Sequencing, Binding Assay

    KLF6 can transactivate and binds to the promoter of TFPI-2 . (A) The p-144 promoter-luciferase activity induced by cotransfection with various amount of KLF6 expression plasmids in MDA-MB-435 cells. The KLF6 cDNA was a generous gift of Scott Friedman and was cloned into PCDNA 3.0 expression vector. (B) Both the predicted KLF6 binding sequence (K6) and the authentic KLF6 binding (Kc) [46] can form specific bands with nuclear extract (NE) from MCF-7 cell lines. And the specific bands were supershifted by the antibody of KLF6. (C) No difference of K6 binding activity was found between MCF-7 and MDA-MB-435 cell lines. Lane 1 to 6: NE from MCF-7; lane 7, 8: NE from MDA-MB-435. Lane 5, 7: 2.5 μg NE; lane 6, 8: 5 μg NE.
    Figure Legend Snippet: KLF6 can transactivate and binds to the promoter of TFPI-2 . (A) The p-144 promoter-luciferase activity induced by cotransfection with various amount of KLF6 expression plasmids in MDA-MB-435 cells. The KLF6 cDNA was a generous gift of Scott Friedman and was cloned into PCDNA 3.0 expression vector. (B) Both the predicted KLF6 binding sequence (K6) and the authentic KLF6 binding (Kc) [46] can form specific bands with nuclear extract (NE) from MCF-7 cell lines. And the specific bands were supershifted by the antibody of KLF6. (C) No difference of K6 binding activity was found between MCF-7 and MDA-MB-435 cell lines. Lane 1 to 6: NE from MCF-7; lane 7, 8: NE from MDA-MB-435. Lane 5, 7: 2.5 μg NE; lane 6, 8: 5 μg NE.

    Techniques Used: Luciferase, Activity Assay, Cotransfection, Expressing, Clone Assay, Plasmid Preparation, Binding Assay, Sequencing

    (A) The sequence of CpG islands in TFPI-2 promoter. The CpG dinucleoide detected by bisulfite modified sequence was indicated in gray shadow. (B) The TFPI-2 gene promoter is highly methylated in MDA-MB-435 cells, whereas it is largely unmethylated in MCF-7 cells. For each cell type, the methylation status of 8 individual clones as determined by bisulfite sequencing analyses is shown in rows 1 to 8. The filled or open circles represent the methylated or unmethylated CpG sites, respectively. (C, D) MDA-MB-435 cells were seeded into 10 cm tissue culture dishes at an initial density of 33% confluence, allowed to attach over night, and then replaced with medium containing 1.25, 2.5 and 5 μM of AZA for 72 hour, then expression of TFPI-2 was detected by western-blot (C) and real-time PCR (D).
    Figure Legend Snippet: (A) The sequence of CpG islands in TFPI-2 promoter. The CpG dinucleoide detected by bisulfite modified sequence was indicated in gray shadow. (B) The TFPI-2 gene promoter is highly methylated in MDA-MB-435 cells, whereas it is largely unmethylated in MCF-7 cells. For each cell type, the methylation status of 8 individual clones as determined by bisulfite sequencing analyses is shown in rows 1 to 8. The filled or open circles represent the methylated or unmethylated CpG sites, respectively. (C, D) MDA-MB-435 cells were seeded into 10 cm tissue culture dishes at an initial density of 33% confluence, allowed to attach over night, and then replaced with medium containing 1.25, 2.5 and 5 μM of AZA for 72 hour, then expression of TFPI-2 was detected by western-blot (C) and real-time PCR (D).

    Techniques Used: Sequencing, Modification, Methylation, Clone Assay, Methylation Sequencing, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    CpG methylation blocked the binding of KLF6 to TFPI-2 promoter . (A) CpG methylation in core matrix decreased the binding ability of KLF6 in vitro. Competition were used to determine the affinity of K6, Kc, methylated K6 (mK6) and muted K6 (Kmute). A 50- fold, 100-fold and 200-fold (50×, 100×, 200×) molar excess of cold K6, Kc, mK6 and Kmute cold probe were chased with labeled Kc. All the sequences were listed in table 1 (B) ChIP reveals that hypermethylation blocks KLF6 binding to the TFPI-2 promoter in vivo. Both CpG Unmethylated MCF-7 and methylated MDA-MB-435 cells were tested for binding of KLF6 by ChIP in vivo. Templates for PCR corresponded to the input used in the ChIP assay (input) and DNA obtained from immunoprecipitations performed in the absence (No antibody) or presence of anti KLF6-specific antibody.
    Figure Legend Snippet: CpG methylation blocked the binding of KLF6 to TFPI-2 promoter . (A) CpG methylation in core matrix decreased the binding ability of KLF6 in vitro. Competition were used to determine the affinity of K6, Kc, methylated K6 (mK6) and muted K6 (Kmute). A 50- fold, 100-fold and 200-fold (50×, 100×, 200×) molar excess of cold K6, Kc, mK6 and Kmute cold probe were chased with labeled Kc. All the sequences were listed in table 1 (B) ChIP reveals that hypermethylation blocks KLF6 binding to the TFPI-2 promoter in vivo. Both CpG Unmethylated MCF-7 and methylated MDA-MB-435 cells were tested for binding of KLF6 by ChIP in vivo. Templates for PCR corresponded to the input used in the ChIP assay (input) and DNA obtained from immunoprecipitations performed in the absence (No antibody) or presence of anti KLF6-specific antibody.

    Techniques Used: CpG Methylation Assay, Binding Assay, In Vitro, Methylation, Labeling, In Vivo

    mda mb 435 breast cancer cell line  (ATCC)


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    ATCC mda mb 435 breast cancer cell line
    Deletion of 5 amino acids from 214 to 218 of IGFBP5 protein promotes <t>MDA-MB-435</t> breast cancer cell motility . A. Representative light-microscopy images from the migration assay. B. Quantification data of in vitro migration assay from vector transfectants (vector 1 and vector 4), wild-type IGFBP5 overexpression clones (wt-c11 and wt-c13), and mutant IGFBP5 overexpression clones (mt-c2, mt-c6, and mt-c29). The error bars represent standard deviations from 2 separate experiments with triplicate samples for each (n = 6). *, p < 0.001. C. Quantification data of in vitro migration assay from transient transfection. The error bars represent standard deviations from triplicate samples. *, p < 0.05 and **, p < 0.001.
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    1) Product Images from "The subcellular localization of IGFBP5 affects its cell growth and migration functions in breast cancer"

    Article Title: The subcellular localization of IGFBP5 affects its cell growth and migration functions in breast cancer

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-9-103

    Deletion of 5 amino acids from 214 to 218 of IGFBP5 protein promotes MDA-MB-435 breast cancer cell motility . A. Representative light-microscopy images from the migration assay. B. Quantification data of in vitro migration assay from vector transfectants (vector 1 and vector 4), wild-type IGFBP5 overexpression clones (wt-c11 and wt-c13), and mutant IGFBP5 overexpression clones (mt-c2, mt-c6, and mt-c29). The error bars represent standard deviations from 2 separate experiments with triplicate samples for each (n = 6). *, p < 0.001. C. Quantification data of in vitro migration assay from transient transfection. The error bars represent standard deviations from triplicate samples. *, p < 0.05 and **, p < 0.001.
    Figure Legend Snippet: Deletion of 5 amino acids from 214 to 218 of IGFBP5 protein promotes MDA-MB-435 breast cancer cell motility . A. Representative light-microscopy images from the migration assay. B. Quantification data of in vitro migration assay from vector transfectants (vector 1 and vector 4), wild-type IGFBP5 overexpression clones (wt-c11 and wt-c13), and mutant IGFBP5 overexpression clones (mt-c2, mt-c6, and mt-c29). The error bars represent standard deviations from 2 separate experiments with triplicate samples for each (n = 6). *, p < 0.001. C. Quantification data of in vitro migration assay from transient transfection. The error bars represent standard deviations from triplicate samples. *, p < 0.05 and **, p < 0.001.

    Techniques Used: Light Microscopy, Migration, In Vitro, Plasmid Preparation, Over Expression, Clone Assay, Mutagenesis, Transfection

    antiproliferation activity against mda mb 435 breast cancer cell lines  (ATCC)


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    ATCC antiproliferation activity against mda mb 435 breast cancer cell lines
    Antiproliferation Activity Against Mda Mb 435 Breast Cancer Cell Lines, 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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    ATCC breast cancer cell line mda mb
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    ATCC human breast cancer cell lines htb129
    <t>HTB129-ctrl</t> and HTB129-miR203 cells were subjected to A) qRT-PCR analyses of SNAI1 mRNA expression in HTB129-ctrl and HTB129-miR203 cells, B) fluorescent staining with DAPI (blue) and phalloidin (red) (scale bar, 20 µm), C) migration and D) invasion assays. E) Predicted miR-203 target sites within SNAI1 3′UTR. F, G) Relative luciferase activity of SNAI1-3′UTR wild type (wt) or mutant (mut) in MDA231 cells transfected with control (F, G), miR-203 (F) or miR-200a/c (G) precursors. Co-transfection with GAPDH 3′UTR vector served as additional negative control (*, p<0.05; **, p<0.01).
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    ATCC mda mb 435s breast cancer cell lines
    A . Experion Bioanalyser gel image for RNA isolated following immunoprecipitation with YB-1 antibody or IgG isotype control antibody (labelled YB-1 IP and IgG IP respectively) from <t>MDA-MB-435S</t> cells. Ribosomal RNAs (18S and 28S) are shown by arrows. The ladder shows the size of the RNAs in nucleotides. Small RNAs are highlighted by the labelled bracket. B . RT-qPCR detection of mRNAs bound following immunoprecipitation with the YB-1 antibody.
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    MiR-27a is significantly increased and CDC27 expression is significantly decreased in <t>TNBC</t> cells. ( A ) qRT-PCR analysis of miR-27a expression in <t>MDA-MB-435,</t> MDA-MB-231, and MCF10A cell lines. ( B ) qRT-PCR analysis of CDC27 mRNA expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. ( C ) Western blot analysis of CDC27 expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. Data are shown as mean±S.D by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.
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    ATCC human breast cancer cell lines mda mb 435
    Expression of TFPI-2 in human breast cancer cells with different metastasis potential . (A). Western blot analysis of TFPI-2 protein expression. The HUVEC cell line was used as positive control and the protein levels of GAPDH were determined as control. (B). Quantitative real-time PCR analysis of TFPI-2 mRNA levels. All expression levels of TFPI-2 in breast cancer cell lines were normalized to the level of its expression in HUVEC. Lane 1: <t>MDA-MB-435.</t> Lane 2: T47D. Lane 3: MCF-7. Lane 4: HUVEC.
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    ATCC mda mb 435 breast cancer cell line
    Deletion of 5 amino acids from 214 to 218 of IGFBP5 protein promotes <t>MDA-MB-435</t> breast cancer cell motility . A. Representative light-microscopy images from the migration assay. B. Quantification data of in vitro migration assay from vector transfectants (vector 1 and vector 4), wild-type IGFBP5 overexpression clones (wt-c11 and wt-c13), and mutant IGFBP5 overexpression clones (mt-c2, mt-c6, and mt-c29). The error bars represent standard deviations from 2 separate experiments with triplicate samples for each (n = 6). *, p < 0.001. C. Quantification data of in vitro migration assay from transient transfection. The error bars represent standard deviations from triplicate samples. *, p < 0.05 and **, p < 0.001.
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    ATCC antiproliferation activity against mda mb 435 breast cancer cell lines
    Deletion of 5 amino acids from 214 to 218 of IGFBP5 protein promotes <t>MDA-MB-435</t> breast cancer cell motility . A. Representative light-microscopy images from the migration assay. B. Quantification data of in vitro migration assay from vector transfectants (vector 1 and vector 4), wild-type IGFBP5 overexpression clones (wt-c11 and wt-c13), and mutant IGFBP5 overexpression clones (mt-c2, mt-c6, and mt-c29). The error bars represent standard deviations from 2 separate experiments with triplicate samples for each (n = 6). *, p < 0.001. C. Quantification data of in vitro migration assay from transient transfection. The error bars represent standard deviations from triplicate samples. *, p < 0.05 and **, p < 0.001.
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    HTB129-ctrl and HTB129-miR203 cells were subjected to A) qRT-PCR analyses of SNAI1 mRNA expression in HTB129-ctrl and HTB129-miR203 cells, B) fluorescent staining with DAPI (blue) and phalloidin (red) (scale bar, 20 µm), C) migration and D) invasion assays. E) Predicted miR-203 target sites within SNAI1 3′UTR. F, G) Relative luciferase activity of SNAI1-3′UTR wild type (wt) or mutant (mut) in MDA231 cells transfected with control (F, G), miR-203 (F) or miR-200a/c (G) precursors. Co-transfection with GAPDH 3′UTR vector served as additional negative control (*, p<0.05; **, p<0.01).

    Journal: PLoS ONE

    Article Title: A Novel Network Integrating a miRNA-203/SNAI1 Feedback Loop which Regulates Epithelial to Mesenchymal Transition

    doi: 10.1371/journal.pone.0035440

    Figure Lengend Snippet: HTB129-ctrl and HTB129-miR203 cells were subjected to A) qRT-PCR analyses of SNAI1 mRNA expression in HTB129-ctrl and HTB129-miR203 cells, B) fluorescent staining with DAPI (blue) and phalloidin (red) (scale bar, 20 µm), C) migration and D) invasion assays. E) Predicted miR-203 target sites within SNAI1 3′UTR. F, G) Relative luciferase activity of SNAI1-3′UTR wild type (wt) or mutant (mut) in MDA231 cells transfected with control (F, G), miR-203 (F) or miR-200a/c (G) precursors. Co-transfection with GAPDH 3′UTR vector served as additional negative control (*, p<0.05; **, p<0.01).

    Article Snippet: The human breast cancer cell lines HTB129 and MDA231 (also named MDA-MB-231 or HTB-26), purchased from the ATCC, were maintained in RPMI1640 and Leibovitz culture media (Lonza), respectively, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin.

    Techniques: Quantitative RT-PCR, Expressing, Staining, Migration, Luciferase, Activity Assay, Mutagenesis, Transfection, Cotransfection, Plasmid Preparation, Negative Control

    A . Experion Bioanalyser gel image for RNA isolated following immunoprecipitation with YB-1 antibody or IgG isotype control antibody (labelled YB-1 IP and IgG IP respectively) from MDA-MB-435S cells. Ribosomal RNAs (18S and 28S) are shown by arrows. The ladder shows the size of the RNAs in nucleotides. Small RNAs are highlighted by the labelled bracket. B . RT-qPCR detection of mRNAs bound following immunoprecipitation with the YB-1 antibody.

    Journal: PLoS ONE

    Article Title: Links between the Oncoprotein YB-1 and Small Non-Coding RNAs in Breast Cancer

    doi: 10.1371/journal.pone.0080171

    Figure Lengend Snippet: A . Experion Bioanalyser gel image for RNA isolated following immunoprecipitation with YB-1 antibody or IgG isotype control antibody (labelled YB-1 IP and IgG IP respectively) from MDA-MB-435S cells. Ribosomal RNAs (18S and 28S) are shown by arrows. The ladder shows the size of the RNAs in nucleotides. Small RNAs are highlighted by the labelled bracket. B . RT-qPCR detection of mRNAs bound following immunoprecipitation with the YB-1 antibody.

    Article Snippet: MCF7 and MDA-MB-435S breast cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA).

    Techniques: Isolation, Immunoprecipitation, Quantitative RT-PCR

    sncRNAs that are bound by YB-1 protein by immunoprecipitation in MCF7 and  MDA-MB-435S  cells based on enrichment in abundance in IP over input.

    Journal: PLoS ONE

    Article Title: Links between the Oncoprotein YB-1 and Small Non-Coding RNAs in Breast Cancer

    doi: 10.1371/journal.pone.0080171

    Figure Lengend Snippet: sncRNAs that are bound by YB-1 protein by immunoprecipitation in MCF7 and MDA-MB-435S cells based on enrichment in abundance in IP over input.

    Article Snippet: MCF7 and MDA-MB-435S breast cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA).

    Techniques: Immunoprecipitation

    A. MCF7 cells B. MDA-MB-435S cells Note: miR-886 abundance in MCF7 cells was undetectable.

    Journal: PLoS ONE

    Article Title: Links between the Oncoprotein YB-1 and Small Non-Coding RNAs in Breast Cancer

    doi: 10.1371/journal.pone.0080171

    Figure Lengend Snippet: A. MCF7 cells B. MDA-MB-435S cells Note: miR-886 abundance in MCF7 cells was undetectable.

    Article Snippet: MCF7 and MDA-MB-435S breast cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA).

    Techniques:

    MiR-27a is significantly increased and CDC27 expression is significantly decreased in TNBC cells. ( A ) qRT-PCR analysis of miR-27a expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. ( B ) qRT-PCR analysis of CDC27 mRNA expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. ( C ) Western blot analysis of CDC27 expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. Data are shown as mean±S.D by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MiR-27a Modulates Radiosensitivity of Triple-Negative Breast Cancer (TNBC) Cells by Targeting CDC27

    doi: 10.12659/MSM.893974

    Figure Lengend Snippet: MiR-27a is significantly increased and CDC27 expression is significantly decreased in TNBC cells. ( A ) qRT-PCR analysis of miR-27a expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. ( B ) qRT-PCR analysis of CDC27 mRNA expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. ( C ) Western blot analysis of CDC27 expression in MDA-MB-435, MDA-MB-231, and MCF10A cell lines. Data are shown as mean±S.D by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

    Article Snippet: Triple negative breast cancer cell line MDA-MB-435 and MDA-MB-231, normal human breast epithelial cell line MCF10A and HEK293T cells were obtained from ATCC.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    MiR-27a is involved in radiosensitivity of TNBC cells. ( A–D ) qRT-PCR analysis of miR-27a expression in MDA-MB-435 ( A, C ) MDA-MB-231 ( B, D ) cells after transfection of 200nM antagomiR-27a ( A, B ) 75nM miR-27a mimics (C, D). ( D–G ) CCK-8 assay of cell proliferation of MDA-MB-435 ( D, F ) MDA-MB-231 ( E, G ) cells transfected with 200 nM antagomiR-27a with/without treatment of IR (8 Gy) (E, F) or transfected with 75 nM miR-27a mimics with/without treatment of IR (8 Gy) ( G, H ). ( I, J ) MDA-MB-435 cells with miR-27a knockdown or overexpression were stained with caspase-3 staining kit 24 h after IR (8 Gy) treatment. Then, flow cytometry analysis was performed to measure the proportion of cells with active caspase-3 staining. Representative flow cytometry images of the cells with active caspase-3 ( I ). Quantification of apoptotic cells with active caspase-3 signal in ( J ). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MiR-27a Modulates Radiosensitivity of Triple-Negative Breast Cancer (TNBC) Cells by Targeting CDC27

    doi: 10.12659/MSM.893974

    Figure Lengend Snippet: MiR-27a is involved in radiosensitivity of TNBC cells. ( A–D ) qRT-PCR analysis of miR-27a expression in MDA-MB-435 ( A, C ) MDA-MB-231 ( B, D ) cells after transfection of 200nM antagomiR-27a ( A, B ) 75nM miR-27a mimics (C, D). ( D–G ) CCK-8 assay of cell proliferation of MDA-MB-435 ( D, F ) MDA-MB-231 ( E, G ) cells transfected with 200 nM antagomiR-27a with/without treatment of IR (8 Gy) (E, F) or transfected with 75 nM miR-27a mimics with/without treatment of IR (8 Gy) ( G, H ). ( I, J ) MDA-MB-435 cells with miR-27a knockdown or overexpression were stained with caspase-3 staining kit 24 h after IR (8 Gy) treatment. Then, flow cytometry analysis was performed to measure the proportion of cells with active caspase-3 staining. Representative flow cytometry images of the cells with active caspase-3 ( I ). Quantification of apoptotic cells with active caspase-3 signal in ( J ). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

    Article Snippet: Triple negative breast cancer cell line MDA-MB-435 and MDA-MB-231, normal human breast epithelial cell line MCF10A and HEK293T cells were obtained from ATCC.

    Techniques: Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Over Expression, Staining, Flow Cytometry

    MiR-27a directly targets CDC27 and regulates its expression in TNBC cells. ( A ) The putative binding site between miR-27a and CDC27 and the designed mutant sequence without the pairing. ( B, C ) The relative firefly luciferase activity in HEK-293T ( B ) and MDA-MB-435 ( C ) cells co-transfected with 150 ng reporter plasmids and 50 nM miR-27a mimics. Both firefly and Renilla luciferase activities were measured 24 h after transfection and the firefly luciferase activity was normalized to the Renilla luciferase activity. ( D, E ) Western blot analysis of CDC27 protein expression in MDA-MB-435 transfected with miR-27a mimics or siCDC-27 ( D ) or transfected with antagomiR-27a or infected with CDC-27 lentiviral particles ( E ). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MiR-27a Modulates Radiosensitivity of Triple-Negative Breast Cancer (TNBC) Cells by Targeting CDC27

    doi: 10.12659/MSM.893974

    Figure Lengend Snippet: MiR-27a directly targets CDC27 and regulates its expression in TNBC cells. ( A ) The putative binding site between miR-27a and CDC27 and the designed mutant sequence without the pairing. ( B, C ) The relative firefly luciferase activity in HEK-293T ( B ) and MDA-MB-435 ( C ) cells co-transfected with 150 ng reporter plasmids and 50 nM miR-27a mimics. Both firefly and Renilla luciferase activities were measured 24 h after transfection and the firefly luciferase activity was normalized to the Renilla luciferase activity. ( D, E ) Western blot analysis of CDC27 protein expression in MDA-MB-435 transfected with miR-27a mimics or siCDC-27 ( D ) or transfected with antagomiR-27a or infected with CDC-27 lentiviral particles ( E ). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

    Article Snippet: Triple negative breast cancer cell line MDA-MB-435 and MDA-MB-231, normal human breast epithelial cell line MCF10A and HEK293T cells were obtained from ATCC.

    Techniques: Expressing, Binding Assay, Mutagenesis, Sequencing, Luciferase, Activity Assay, Transfection, Western Blot, Infection

    MiR-27a modulates radiosensitivity of TNBC cells through targeting CDC27. ( A–D ) CCK-8 assay of cell proliferation in MDA-MB-435 ( A, C ) MDA-MB-231 ( B, D ) cells after infection with CDC27 lentiviral particle with/without treatment of IR (8 Gy) ( A, B ) or transfected with 50 nM siCDC27 with/without treatment of IR (8 Gy) ( C, D ). ( E, F ) The effect of co-transfection of miR-27a and CDC27 on cell proliferation in MDA-MB-435 ( E ) MDA-MB-231 ( F ) cells with/without treatment of IR (8 Gy). ( G, H ) The effect of co-transfection of antagomiR-27a and siCDC27 on cell proliferation in MDA-MB-435 ( G ) MDA-MB-231 ( H ) cells with/without treatment of IR (8 Gy). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MiR-27a Modulates Radiosensitivity of Triple-Negative Breast Cancer (TNBC) Cells by Targeting CDC27

    doi: 10.12659/MSM.893974

    Figure Lengend Snippet: MiR-27a modulates radiosensitivity of TNBC cells through targeting CDC27. ( A–D ) CCK-8 assay of cell proliferation in MDA-MB-435 ( A, C ) MDA-MB-231 ( B, D ) cells after infection with CDC27 lentiviral particle with/without treatment of IR (8 Gy) ( A, B ) or transfected with 50 nM siCDC27 with/without treatment of IR (8 Gy) ( C, D ). ( E, F ) The effect of co-transfection of miR-27a and CDC27 on cell proliferation in MDA-MB-435 ( E ) MDA-MB-231 ( F ) cells with/without treatment of IR (8 Gy). ( G, H ) The effect of co-transfection of antagomiR-27a and siCDC27 on cell proliferation in MDA-MB-435 ( G ) MDA-MB-231 ( H ) cells with/without treatment of IR (8 Gy). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

    Article Snippet: Triple negative breast cancer cell line MDA-MB-435 and MDA-MB-231, normal human breast epithelial cell line MCF10A and HEK293T cells were obtained from ATCC.

    Techniques: CCK-8 Assay, Infection, Transfection, Cotransfection

    Expression of TFPI-2 in human breast cancer cells with different metastasis potential . (A). Western blot analysis of TFPI-2 protein expression. The HUVEC cell line was used as positive control and the protein levels of GAPDH were determined as control. (B). Quantitative real-time PCR analysis of TFPI-2 mRNA levels. All expression levels of TFPI-2 in breast cancer cell lines were normalized to the level of its expression in HUVEC. Lane 1: MDA-MB-435. Lane 2: T47D. Lane 3: MCF-7. Lane 4: HUVEC.

    Journal: BMC Molecular Biology

    Article Title: Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells

    doi: 10.1186/1471-2199-8-110

    Figure Lengend Snippet: Expression of TFPI-2 in human breast cancer cells with different metastasis potential . (A). Western blot analysis of TFPI-2 protein expression. The HUVEC cell line was used as positive control and the protein levels of GAPDH were determined as control. (B). Quantitative real-time PCR analysis of TFPI-2 mRNA levels. All expression levels of TFPI-2 in breast cancer cell lines were normalized to the level of its expression in HUVEC. Lane 1: MDA-MB-435. Lane 2: T47D. Lane 3: MCF-7. Lane 4: HUVEC.

    Article Snippet: Human breast cancer cell lines MDA-MB-435, MDA-MB-231, T47D and MCF-7 were purchased from American Type Culture collection (ATCC, Manassas, VA), cultured in DMEM with 5% CO 2 .

    Techniques: Expressing, Western Blot, Positive Control, Real-time Polymerase Chain Reaction

    Analysis of TFPI-2 promoter . (A) Breast cancer cell lines were transfected with promoter-luciferase plasmid cloned from different cell lines, bearing respective genetic mutation. The promoter from different cell line has nearly the same luciferase activity in both MDA-MB-435 and MCF-7 cell lines. Results are shown as mean ± SD. The apparent lower level of promoter activity in MDA-MB-435 cell was not statistically significant (p = 0.342>0.05). (B) Luciferase reporter gene constructs with 5'ends between nucleotides -1436 and -144 and a common 3'end at +75 were transiently transfected into breast cancer cells. The minimal construct P-144 has the same luciferase activity as P-1436. (C) Linker-scan mutation analysis of TFPI-2 promoter -144 to +1 region. The fragment from -144 to the transcriptional start point (+1) of TFPI-2 promoter were systematically replaced with an BamH I & Xba I polylinker (GGATCCTCTAGA), the resulting sequence were analyzed for not introducing new transcription factor binding site by MatInspector [21]. SM1 indicate the promoter sequence from -12 to +1, and in turn every 12 bases from -144 to -12 was indicated by SM12 to SM2. Then the luciferase promoter reporter plasmids were transfected into MDA-MB-435 cell line and the reported luciferase activity was measured. Results are shown as mean ± SD relative to the wild type (*p < 0.05; **p < 0.001).

    Journal: BMC Molecular Biology

    Article Title: Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells

    doi: 10.1186/1471-2199-8-110

    Figure Lengend Snippet: Analysis of TFPI-2 promoter . (A) Breast cancer cell lines were transfected with promoter-luciferase plasmid cloned from different cell lines, bearing respective genetic mutation. The promoter from different cell line has nearly the same luciferase activity in both MDA-MB-435 and MCF-7 cell lines. Results are shown as mean ± SD. The apparent lower level of promoter activity in MDA-MB-435 cell was not statistically significant (p = 0.342>0.05). (B) Luciferase reporter gene constructs with 5'ends between nucleotides -1436 and -144 and a common 3'end at +75 were transiently transfected into breast cancer cells. The minimal construct P-144 has the same luciferase activity as P-1436. (C) Linker-scan mutation analysis of TFPI-2 promoter -144 to +1 region. The fragment from -144 to the transcriptional start point (+1) of TFPI-2 promoter were systematically replaced with an BamH I & Xba I polylinker (GGATCCTCTAGA), the resulting sequence were analyzed for not introducing new transcription factor binding site by MatInspector [21]. SM1 indicate the promoter sequence from -12 to +1, and in turn every 12 bases from -144 to -12 was indicated by SM12 to SM2. Then the luciferase promoter reporter plasmids were transfected into MDA-MB-435 cell line and the reported luciferase activity was measured. Results are shown as mean ± SD relative to the wild type (*p < 0.05; **p < 0.001).

    Article Snippet: Human breast cancer cell lines MDA-MB-435, MDA-MB-231, T47D and MCF-7 were purchased from American Type Culture collection (ATCC, Manassas, VA), cultured in DMEM with 5% CO 2 .

    Techniques: Transfection, Luciferase, Plasmid Preparation, Clone Assay, Mutagenesis, Activity Assay, Construct, Sequencing, Binding Assay

    KLF6 can transactivate and binds to the promoter of TFPI-2 . (A) The p-144 promoter-luciferase activity induced by cotransfection with various amount of KLF6 expression plasmids in MDA-MB-435 cells. The KLF6 cDNA was a generous gift of Scott Friedman and was cloned into PCDNA 3.0 expression vector. (B) Both the predicted KLF6 binding sequence (K6) and the authentic KLF6 binding (Kc) [46] can form specific bands with nuclear extract (NE) from MCF-7 cell lines. And the specific bands were supershifted by the antibody of KLF6. (C) No difference of K6 binding activity was found between MCF-7 and MDA-MB-435 cell lines. Lane 1 to 6: NE from MCF-7; lane 7, 8: NE from MDA-MB-435. Lane 5, 7: 2.5 μg NE; lane 6, 8: 5 μg NE.

    Journal: BMC Molecular Biology

    Article Title: Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells

    doi: 10.1186/1471-2199-8-110

    Figure Lengend Snippet: KLF6 can transactivate and binds to the promoter of TFPI-2 . (A) The p-144 promoter-luciferase activity induced by cotransfection with various amount of KLF6 expression plasmids in MDA-MB-435 cells. The KLF6 cDNA was a generous gift of Scott Friedman and was cloned into PCDNA 3.0 expression vector. (B) Both the predicted KLF6 binding sequence (K6) and the authentic KLF6 binding (Kc) [46] can form specific bands with nuclear extract (NE) from MCF-7 cell lines. And the specific bands were supershifted by the antibody of KLF6. (C) No difference of K6 binding activity was found between MCF-7 and MDA-MB-435 cell lines. Lane 1 to 6: NE from MCF-7; lane 7, 8: NE from MDA-MB-435. Lane 5, 7: 2.5 μg NE; lane 6, 8: 5 μg NE.

    Article Snippet: Human breast cancer cell lines MDA-MB-435, MDA-MB-231, T47D and MCF-7 were purchased from American Type Culture collection (ATCC, Manassas, VA), cultured in DMEM with 5% CO 2 .

    Techniques: Luciferase, Activity Assay, Cotransfection, Expressing, Clone Assay, Plasmid Preparation, Binding Assay, Sequencing

    (A) The sequence of CpG islands in TFPI-2 promoter. The CpG dinucleoide detected by bisulfite modified sequence was indicated in gray shadow. (B) The TFPI-2 gene promoter is highly methylated in MDA-MB-435 cells, whereas it is largely unmethylated in MCF-7 cells. For each cell type, the methylation status of 8 individual clones as determined by bisulfite sequencing analyses is shown in rows 1 to 8. The filled or open circles represent the methylated or unmethylated CpG sites, respectively. (C, D) MDA-MB-435 cells were seeded into 10 cm tissue culture dishes at an initial density of 33% confluence, allowed to attach over night, and then replaced with medium containing 1.25, 2.5 and 5 μM of AZA for 72 hour, then expression of TFPI-2 was detected by western-blot (C) and real-time PCR (D).

    Journal: BMC Molecular Biology

    Article Title: Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells

    doi: 10.1186/1471-2199-8-110

    Figure Lengend Snippet: (A) The sequence of CpG islands in TFPI-2 promoter. The CpG dinucleoide detected by bisulfite modified sequence was indicated in gray shadow. (B) The TFPI-2 gene promoter is highly methylated in MDA-MB-435 cells, whereas it is largely unmethylated in MCF-7 cells. For each cell type, the methylation status of 8 individual clones as determined by bisulfite sequencing analyses is shown in rows 1 to 8. The filled or open circles represent the methylated or unmethylated CpG sites, respectively. (C, D) MDA-MB-435 cells were seeded into 10 cm tissue culture dishes at an initial density of 33% confluence, allowed to attach over night, and then replaced with medium containing 1.25, 2.5 and 5 μM of AZA for 72 hour, then expression of TFPI-2 was detected by western-blot (C) and real-time PCR (D).

    Article Snippet: Human breast cancer cell lines MDA-MB-435, MDA-MB-231, T47D and MCF-7 were purchased from American Type Culture collection (ATCC, Manassas, VA), cultured in DMEM with 5% CO 2 .

    Techniques: Sequencing, Modification, Methylation, Clone Assay, Methylation Sequencing, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    CpG methylation blocked the binding of KLF6 to TFPI-2 promoter . (A) CpG methylation in core matrix decreased the binding ability of KLF6 in vitro. Competition were used to determine the affinity of K6, Kc, methylated K6 (mK6) and muted K6 (Kmute). A 50- fold, 100-fold and 200-fold (50×, 100×, 200×) molar excess of cold K6, Kc, mK6 and Kmute cold probe were chased with labeled Kc. All the sequences were listed in table 1 (B) ChIP reveals that hypermethylation blocks KLF6 binding to the TFPI-2 promoter in vivo. Both CpG Unmethylated MCF-7 and methylated MDA-MB-435 cells were tested for binding of KLF6 by ChIP in vivo. Templates for PCR corresponded to the input used in the ChIP assay (input) and DNA obtained from immunoprecipitations performed in the absence (No antibody) or presence of anti KLF6-specific antibody.

    Journal: BMC Molecular Biology

    Article Title: Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells

    doi: 10.1186/1471-2199-8-110

    Figure Lengend Snippet: CpG methylation blocked the binding of KLF6 to TFPI-2 promoter . (A) CpG methylation in core matrix decreased the binding ability of KLF6 in vitro. Competition were used to determine the affinity of K6, Kc, methylated K6 (mK6) and muted K6 (Kmute). A 50- fold, 100-fold and 200-fold (50×, 100×, 200×) molar excess of cold K6, Kc, mK6 and Kmute cold probe were chased with labeled Kc. All the sequences were listed in table 1 (B) ChIP reveals that hypermethylation blocks KLF6 binding to the TFPI-2 promoter in vivo. Both CpG Unmethylated MCF-7 and methylated MDA-MB-435 cells were tested for binding of KLF6 by ChIP in vivo. Templates for PCR corresponded to the input used in the ChIP assay (input) and DNA obtained from immunoprecipitations performed in the absence (No antibody) or presence of anti KLF6-specific antibody.

    Article Snippet: Human breast cancer cell lines MDA-MB-435, MDA-MB-231, T47D and MCF-7 were purchased from American Type Culture collection (ATCC, Manassas, VA), cultured in DMEM with 5% CO 2 .

    Techniques: CpG Methylation Assay, Binding Assay, In Vitro, Methylation, Labeling, In Vivo

    Deletion of 5 amino acids from 214 to 218 of IGFBP5 protein promotes MDA-MB-435 breast cancer cell motility . A. Representative light-microscopy images from the migration assay. B. Quantification data of in vitro migration assay from vector transfectants (vector 1 and vector 4), wild-type IGFBP5 overexpression clones (wt-c11 and wt-c13), and mutant IGFBP5 overexpression clones (mt-c2, mt-c6, and mt-c29). The error bars represent standard deviations from 2 separate experiments with triplicate samples for each (n = 6). *, p < 0.001. C. Quantification data of in vitro migration assay from transient transfection. The error bars represent standard deviations from triplicate samples. *, p < 0.05 and **, p < 0.001.

    Journal: BMC Cancer

    Article Title: The subcellular localization of IGFBP5 affects its cell growth and migration functions in breast cancer

    doi: 10.1186/1471-2407-9-103

    Figure Lengend Snippet: Deletion of 5 amino acids from 214 to 218 of IGFBP5 protein promotes MDA-MB-435 breast cancer cell motility . A. Representative light-microscopy images from the migration assay. B. Quantification data of in vitro migration assay from vector transfectants (vector 1 and vector 4), wild-type IGFBP5 overexpression clones (wt-c11 and wt-c13), and mutant IGFBP5 overexpression clones (mt-c2, mt-c6, and mt-c29). The error bars represent standard deviations from 2 separate experiments with triplicate samples for each (n = 6). *, p < 0.001. C. Quantification data of in vitro migration assay from transient transfection. The error bars represent standard deviations from triplicate samples. *, p < 0.05 and **, p < 0.001.

    Article Snippet: The MDA-MB-435 breast cancer cell line was obtained from the American Type Culture Company (ATCC; Menassas, VA) and was routinely maintained in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic cocktail under standard conditions.

    Techniques: Light Microscopy, Migration, In Vitro, Plasmid Preparation, Over Expression, Clone Assay, Mutagenesis, Transfection