bt 549 cells  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH bt 549 cells
    a , b Immunofluorescence images of A549 cells ( a ) and <t>BT-549</t> ( b ) showing dsRNA-specific J2 signals in hnRNPK KD cells. The panel to the right shows the merged images. Magnified J2 signals (puncta) are shown in an inset at the upper right corner indicated by white box. Indicative scale bar is 50um. Graphs show the number of J2 signals in hnRNPK KD cells quantified using Imaris spot detection tools. For A549 cell line - control: n = 59, siRNA1: n = 64 and siRNA2: n = 73 cells and for BT-549: control: n = 25, siRNA1: n = 37 and siRNA2: n = 44 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. Significance was calculated by One-way ANOVA (ordinary multiple comparisons test). c , e RNAscope images of A549 cells ( c ) and BT-549 cells ( e ) showing IER3 (red) and IER3-AS1 (green) in control and hnRNPK KD cells. Panel to the right shows the magnified images of the merged panels indicated by white box. White arrows marks IER3-AS1 (green dots) and yellow arrows marks colocalized IER3/IER3-AS1 dsRNA (Yellow dots). Indicative scale bar on the images is 50 um. Bar graph shows the number of IER3-AS1 and IER3 RNA signals per cell in control and hnRNPK KD cells quantified using Imaris spot detection tools. For A549 - control: n = 101, siRNA1: n = 78 and siRNA2: n = 97 cells and for BT-549 - control: n = 132, siRNA1: n = 44 and siRNA2: n = F36 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. The significance was calculated by Two-way ANOVA (Sidak’s multiple comparisons test). d Immunoblot showing the levels of HnRNPK and IER3 in hnRNPK KD A549 cells. GAPDH was used as an internal loading control. Similar results were observed in an independent biological replicate. Histograms showing the relative band intensities from the western blot are shown left side. The p values were calculated using two-sided Student’s t -test and data are presented ± SD from two independent replicates.
    Bt 549 Cells, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "HnRNPK maintains single strand RNA through controlling double-strand RNA in mammalian cells"

    Article Title: HnRNPK maintains single strand RNA through controlling double-strand RNA in mammalian cells

    Journal: Nature Communications

    doi: 10.1038/s41467-022-32537-0

    a , b Immunofluorescence images of A549 cells ( a ) and BT-549 ( b ) showing dsRNA-specific J2 signals in hnRNPK KD cells. The panel to the right shows the merged images. Magnified J2 signals (puncta) are shown in an inset at the upper right corner indicated by white box. Indicative scale bar is 50um. Graphs show the number of J2 signals in hnRNPK KD cells quantified using Imaris spot detection tools. For A549 cell line - control: n = 59, siRNA1: n = 64 and siRNA2: n = 73 cells and for BT-549: control: n = 25, siRNA1: n = 37 and siRNA2: n = 44 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. Significance was calculated by One-way ANOVA (ordinary multiple comparisons test). c , e RNAscope images of A549 cells ( c ) and BT-549 cells ( e ) showing IER3 (red) and IER3-AS1 (green) in control and hnRNPK KD cells. Panel to the right shows the magnified images of the merged panels indicated by white box. White arrows marks IER3-AS1 (green dots) and yellow arrows marks colocalized IER3/IER3-AS1 dsRNA (Yellow dots). Indicative scale bar on the images is 50 um. Bar graph shows the number of IER3-AS1 and IER3 RNA signals per cell in control and hnRNPK KD cells quantified using Imaris spot detection tools. For A549 - control: n = 101, siRNA1: n = 78 and siRNA2: n = 97 cells and for BT-549 - control: n = 132, siRNA1: n = 44 and siRNA2: n = F36 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. The significance was calculated by Two-way ANOVA (Sidak’s multiple comparisons test). d Immunoblot showing the levels of HnRNPK and IER3 in hnRNPK KD A549 cells. GAPDH was used as an internal loading control. Similar results were observed in an independent biological replicate. Histograms showing the relative band intensities from the western blot are shown left side. The p values were calculated using two-sided Student’s t -test and data are presented ± SD from two independent replicates.
    Figure Legend Snippet: a , b Immunofluorescence images of A549 cells ( a ) and BT-549 ( b ) showing dsRNA-specific J2 signals in hnRNPK KD cells. The panel to the right shows the merged images. Magnified J2 signals (puncta) are shown in an inset at the upper right corner indicated by white box. Indicative scale bar is 50um. Graphs show the number of J2 signals in hnRNPK KD cells quantified using Imaris spot detection tools. For A549 cell line - control: n = 59, siRNA1: n = 64 and siRNA2: n = 73 cells and for BT-549: control: n = 25, siRNA1: n = 37 and siRNA2: n = 44 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. Significance was calculated by One-way ANOVA (ordinary multiple comparisons test). c , e RNAscope images of A549 cells ( c ) and BT-549 cells ( e ) showing IER3 (red) and IER3-AS1 (green) in control and hnRNPK KD cells. Panel to the right shows the magnified images of the merged panels indicated by white box. White arrows marks IER3-AS1 (green dots) and yellow arrows marks colocalized IER3/IER3-AS1 dsRNA (Yellow dots). Indicative scale bar on the images is 50 um. Bar graph shows the number of IER3-AS1 and IER3 RNA signals per cell in control and hnRNPK KD cells quantified using Imaris spot detection tools. For A549 - control: n = 101, siRNA1: n = 78 and siRNA2: n = 97 cells and for BT-549 - control: n = 132, siRNA1: n = 44 and siRNA2: n = F36 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. The significance was calculated by Two-way ANOVA (Sidak’s multiple comparisons test). d Immunoblot showing the levels of HnRNPK and IER3 in hnRNPK KD A549 cells. GAPDH was used as an internal loading control. Similar results were observed in an independent biological replicate. Histograms showing the relative band intensities from the western blot are shown left side. The p values were calculated using two-sided Student’s t -test and data are presented ± SD from two independent replicates.

    Techniques Used: Immunofluorescence, Western Blot

    bt549  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH bt549
    Bt549, supplied by CLS Cell Lines Service GmbH, 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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    bt 549 cells  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH bt 549 cells
    a , b Immunofluorescence images of A549 cells ( a ) and <t>BT-549</t> ( b ) showing dsRNA-specific J2 signals in hnRNPK KD cells. The panel to the right shows the merged images. Magnified J2 signals (puncta) are shown in an inset at the upper right corner indicated by white box. Indicative scale bar is 50um. Graphs show the number of J2 signals in hnRNPK KD cells quantified using Imaris spot detection tools. For A549 cell line - control: n = 59, siRNA1: n = 64 and siRNA2: n = 73 cells and for BT-549: control: n = 25, siRNA1: n = 37 and siRNA2: n = 44 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. Significance was calculated by One-way ANOVA (ordinary multiple comparisons test). c , e RNAscope images of A549 cells ( c ) and BT-549 cells ( e ) showing IER3 (red) and IER3-AS1 (green) in control and hnRNPK KD cells. Panel to the right shows the magnified images of the merged panels indicated by white box. White arrows marks IER3-AS1 (green dots) and yellow arrows marks colocalized IER3/IER3-AS1 dsRNA (Yellow dots). Indicative scale bar on the images is 50 um. Bar graph shows the number of IER3-AS1 and IER3 RNA signals per cell in control and hnRNPK KD cells quantified using Imaris spot detection tools. For A549 - control: n = 101, siRNA1: n = 78 and siRNA2: n = 97 cells and for BT-549 - control: n = 132, siRNA1: n = 44 and siRNA2: n = F36 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. The significance was calculated by Two-way ANOVA (Sidak’s multiple comparisons test). d Immunoblot showing the levels of HnRNPK and IER3 in hnRNPK KD A549 cells. GAPDH was used as an internal loading control. Similar results were observed in an independent biological replicate. Histograms showing the relative band intensities from the western blot are shown left side. The p values were calculated using two-sided Student’s t -test and data are presented ± SD from two independent replicates.
    Bt 549 Cells, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "HnRNPK maintains single strand RNA through controlling double-strand RNA in mammalian cells"

    Article Title: HnRNPK maintains single strand RNA through controlling double-strand RNA in mammalian cells

    Journal: Nature Communications

    doi: 10.1038/s41467-022-32537-0

    a , b Immunofluorescence images of A549 cells ( a ) and BT-549 ( b ) showing dsRNA-specific J2 signals in hnRNPK KD cells. The panel to the right shows the merged images. Magnified J2 signals (puncta) are shown in an inset at the upper right corner indicated by white box. Indicative scale bar is 50um. Graphs show the number of J2 signals in hnRNPK KD cells quantified using Imaris spot detection tools. For A549 cell line - control: n = 59, siRNA1: n = 64 and siRNA2: n = 73 cells and for BT-549: control: n = 25, siRNA1: n = 37 and siRNA2: n = 44 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. Significance was calculated by One-way ANOVA (ordinary multiple comparisons test). c , e RNAscope images of A549 cells ( c ) and BT-549 cells ( e ) showing IER3 (red) and IER3-AS1 (green) in control and hnRNPK KD cells. Panel to the right shows the magnified images of the merged panels indicated by white box. White arrows marks IER3-AS1 (green dots) and yellow arrows marks colocalized IER3/IER3-AS1 dsRNA (Yellow dots). Indicative scale bar on the images is 50 um. Bar graph shows the number of IER3-AS1 and IER3 RNA signals per cell in control and hnRNPK KD cells quantified using Imaris spot detection tools. For A549 - control: n = 101, siRNA1: n = 78 and siRNA2: n = 97 cells and for BT-549 - control: n = 132, siRNA1: n = 44 and siRNA2: n = F36 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. The significance was calculated by Two-way ANOVA (Sidak’s multiple comparisons test). d Immunoblot showing the levels of HnRNPK and IER3 in hnRNPK KD A549 cells. GAPDH was used as an internal loading control. Similar results were observed in an independent biological replicate. Histograms showing the relative band intensities from the western blot are shown left side. The p values were calculated using two-sided Student’s t -test and data are presented ± SD from two independent replicates.
    Figure Legend Snippet: a , b Immunofluorescence images of A549 cells ( a ) and BT-549 ( b ) showing dsRNA-specific J2 signals in hnRNPK KD cells. The panel to the right shows the merged images. Magnified J2 signals (puncta) are shown in an inset at the upper right corner indicated by white box. Indicative scale bar is 50um. Graphs show the number of J2 signals in hnRNPK KD cells quantified using Imaris spot detection tools. For A549 cell line - control: n = 59, siRNA1: n = 64 and siRNA2: n = 73 cells and for BT-549: control: n = 25, siRNA1: n = 37 and siRNA2: n = 44 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. Significance was calculated by One-way ANOVA (ordinary multiple comparisons test). c , e RNAscope images of A549 cells ( c ) and BT-549 cells ( e ) showing IER3 (red) and IER3-AS1 (green) in control and hnRNPK KD cells. Panel to the right shows the magnified images of the merged panels indicated by white box. White arrows marks IER3-AS1 (green dots) and yellow arrows marks colocalized IER3/IER3-AS1 dsRNA (Yellow dots). Indicative scale bar on the images is 50 um. Bar graph shows the number of IER3-AS1 and IER3 RNA signals per cell in control and hnRNPK KD cells quantified using Imaris spot detection tools. For A549 - control: n = 101, siRNA1: n = 78 and siRNA2: n = 97 cells and for BT-549 - control: n = 132, siRNA1: n = 44 and siRNA2: n = F36 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. The significance was calculated by Two-way ANOVA (Sidak’s multiple comparisons test). d Immunoblot showing the levels of HnRNPK and IER3 in hnRNPK KD A549 cells. GAPDH was used as an internal loading control. Similar results were observed in an independent biological replicate. Histograms showing the relative band intensities from the western blot are shown left side. The p values were calculated using two-sided Student’s t -test and data are presented ± SD from two independent replicates.

    Techniques Used: Immunofluorescence, Western Blot

    humantnbc cells  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH humantnbc cells
    Humantnbc Cells, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bt549  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH bt549
    Bt549, supplied by CLS Cell Lines Service GmbH, 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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    bt549  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH bt549
    Bt549, supplied by CLS Cell Lines Service GmbH, 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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    bt549  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH bt549
    Bt549, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bt549  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH bt549
    UPAR expression in bladder and breast cancer cell lines. Bladder and breast cancer cell lines resembling respectively different grades (RT4 -grade 1-, RT112 and 5637- grade 2-, HT1376 and ECV304 -grade 3-) and subtypes (MDA-MB-468, <t>BT549,</t> SUM149, SUM159 -TNBC-, SKBR3 -HER2 + -) were analyzed by quantitative PCR (see “Materials and Methods”) for UPAR gene expression ( A ) and by flow cytometry for uPAR protein expression (b and c). UPAR protein expression is shown as histogram plots ( B ) and Relative Fluorescence Intensity (RFI) (see “Materials and Methods”) ( C ). SKBR3 HER2 + breast cancer cell line was used as subtype control. The dashed line represents the threshold arbitrarily defining positive expression (RFI = 2).
    Bt549, supplied by CLS Cell Lines Service GmbH, 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 "The anti-tumoral potential of the saporin-based uPAR-targeting chimera ATF-SAP"

    Article Title: The anti-tumoral potential of the saporin-based uPAR-targeting chimera ATF-SAP

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-59313-8

    UPAR expression in bladder and breast cancer cell lines. Bladder and breast cancer cell lines resembling respectively different grades (RT4 -grade 1-, RT112 and 5637- grade 2-, HT1376 and ECV304 -grade 3-) and subtypes (MDA-MB-468, BT549, SUM149, SUM159 -TNBC-, SKBR3 -HER2 + -) were analyzed by quantitative PCR (see “Materials and Methods”) for UPAR gene expression ( A ) and by flow cytometry for uPAR protein expression (b and c). UPAR protein expression is shown as histogram plots ( B ) and Relative Fluorescence Intensity (RFI) (see “Materials and Methods”) ( C ). SKBR3 HER2 + breast cancer cell line was used as subtype control. The dashed line represents the threshold arbitrarily defining positive expression (RFI = 2).
    Figure Legend Snippet: UPAR expression in bladder and breast cancer cell lines. Bladder and breast cancer cell lines resembling respectively different grades (RT4 -grade 1-, RT112 and 5637- grade 2-, HT1376 and ECV304 -grade 3-) and subtypes (MDA-MB-468, BT549, SUM149, SUM159 -TNBC-, SKBR3 -HER2 + -) were analyzed by quantitative PCR (see “Materials and Methods”) for UPAR gene expression ( A ) and by flow cytometry for uPAR protein expression (b and c). UPAR protein expression is shown as histogram plots ( B ) and Relative Fluorescence Intensity (RFI) (see “Materials and Methods”) ( C ). SKBR3 HER2 + breast cancer cell line was used as subtype control. The dashed line represents the threshold arbitrarily defining positive expression (RFI = 2).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Fluorescence

    Cytotoxic activity of ATF-SAP on bladder and breast cancer cells. ATF-SAP activity and target specificity were evaluated on RT4, RT112, 5637, HT1376 and ECV304 bladder cancer cell lines ( A ) and on MDA-MB-468, SUM149, SUM159, BT549 TNBC and HER2 + SKBR3 breast cancer cell lines. ( B ) Cells were incubated for 72 h with scalar logarithmic concentrations of the toxin and cell viability was analyzed by MTT assay. The untargeted seed SAP and the catalytically inactive mutant ATF-SAP KQ were used as controls. The IC50 from three different experiments is reported as mean ± SE.
    Figure Legend Snippet: Cytotoxic activity of ATF-SAP on bladder and breast cancer cells. ATF-SAP activity and target specificity were evaluated on RT4, RT112, 5637, HT1376 and ECV304 bladder cancer cell lines ( A ) and on MDA-MB-468, SUM149, SUM159, BT549 TNBC and HER2 + SKBR3 breast cancer cell lines. ( B ) Cells were incubated for 72 h with scalar logarithmic concentrations of the toxin and cell viability was analyzed by MTT assay. The untargeted seed SAP and the catalytically inactive mutant ATF-SAP KQ were used as controls. The IC50 from three different experiments is reported as mean ± SE.

    Techniques Used: Activity Assay, Incubation, MTT Assay, Mutagenesis

    UPA and LRP1 expression in bladder and breast cancer cell lines. ( A ) Skin fibroblasts, bladder and breast cancer cell lines respectively resembling different stages (RT4 -T1 superficial-, RT112 and 5637- T2 muscle invasive-, HT1376 and ECV304 -T3 muscle invasive-) and subtypes (MDA-MB-468, MDA-MB-231, BT549, SUM149, SUM159 -TNBC-, SKBR3 -HER2 + -) were analyzed by qPCR for UPA gene expression (see “Materials and Methods”). ( B ) Competition assay between ATF-SAP and uPAR natural ligands. MDA-MB-468 cells were incubated with ATF-SAP 20 nM in the presence of equal or increasing concentrations of uPA or PAI. The effect on cell viability was evaluated after 72 h by MTT assay. Seed SAP was used as untargeted control. ( C ) Comparison of pro-uPA-SAP and ATF-SAP toxic activity on fibroblasts and MDA-MB-231 cells. Results from one representative experiment are shown as mean ± SD. Three independent experiments were performed for each assay. LRP1 gene ( D ) and protein ( E ) expression on bladder and breast cancer cells. LRP1 protein expression is displayed as histogram plots ( E , left panel) and RFI (see “Materials and Methods”) ( E , right panel). The dashed line represents the threshold arbitrarily defining positive expression (RFI = 2).
    Figure Legend Snippet: UPA and LRP1 expression in bladder and breast cancer cell lines. ( A ) Skin fibroblasts, bladder and breast cancer cell lines respectively resembling different stages (RT4 -T1 superficial-, RT112 and 5637- T2 muscle invasive-, HT1376 and ECV304 -T3 muscle invasive-) and subtypes (MDA-MB-468, MDA-MB-231, BT549, SUM149, SUM159 -TNBC-, SKBR3 -HER2 + -) were analyzed by qPCR for UPA gene expression (see “Materials and Methods”). ( B ) Competition assay between ATF-SAP and uPAR natural ligands. MDA-MB-468 cells were incubated with ATF-SAP 20 nM in the presence of equal or increasing concentrations of uPA or PAI. The effect on cell viability was evaluated after 72 h by MTT assay. Seed SAP was used as untargeted control. ( C ) Comparison of pro-uPA-SAP and ATF-SAP toxic activity on fibroblasts and MDA-MB-231 cells. Results from one representative experiment are shown as mean ± SD. Three independent experiments were performed for each assay. LRP1 gene ( D ) and protein ( E ) expression on bladder and breast cancer cells. LRP1 protein expression is displayed as histogram plots ( E , left panel) and RFI (see “Materials and Methods”) ( E , right panel). The dashed line represents the threshold arbitrarily defining positive expression (RFI = 2).

    Techniques Used: Expressing, Competitive Binding Assay, Incubation, MTT Assay, Activity Assay

    bt 549  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH bt 549
    Characterizing H3K4me2 enriched lncRNAs. ( A ) ChRIP experimental workflow to identify lncRNAs bound to chromatin enriched with H3K4me2 and WDR5 in <t>BT-549</t> cell line. ( B ) Computational approach used for finding chromatin-associated RNAs and enriched patterns of genomic organization with respect to nearest protein coding genes. ( C ) Scatter plot showing the enrichment of H3K4me2 lncRNAs over input. X-axis denotes log transformed expression values of H3K4me2 lncRNAs and Y-axis denotes log-fold changes of lncRNAs in H3K4me2 ChRIP sample over nuclear input sample. ( D ) Histogram shows distribution of H3K4me2 lncRNAs with antisense (X) and sense (S) pattern with respect to their protein coding partner (pPCGs). ‘n’ denotes number of lncRNA-pPCG pairs in each pattern. ( E ) Genomic organization of H3K4me2 lncRNAs (green bars) with respect to nearest protein coding genes (grey bars). XH: where lncRNA shows Head-to-Head arrangement with nearby protein coding gene. XT: lncRNA in Tail-to-Tail arrangement with protein coding gene, XI: lncRNA located inside a protein coding gene, XO: lncRNA located outside and covers the entire protein coding gene. Sense pairs means lncRNAs in the same orientation as protein coding gene. The notion of greater or less than 2 kb means that the partner genes (pPCGs) are located within 2 kb (<2 kb) or away from 2 kb (> 2 kb) but within 50 kb window with respect to H3K4me2 lncRNAs. ( F ) Distribution of different patterns of genomic arrangements as described in (E) for H3K4me2 lncRNAs and non-CAR lncRNAs.
    Bt 549, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units"

    Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky635

    Characterizing H3K4me2 enriched lncRNAs. ( A ) ChRIP experimental workflow to identify lncRNAs bound to chromatin enriched with H3K4me2 and WDR5 in BT-549 cell line. ( B ) Computational approach used for finding chromatin-associated RNAs and enriched patterns of genomic organization with respect to nearest protein coding genes. ( C ) Scatter plot showing the enrichment of H3K4me2 lncRNAs over input. X-axis denotes log transformed expression values of H3K4me2 lncRNAs and Y-axis denotes log-fold changes of lncRNAs in H3K4me2 ChRIP sample over nuclear input sample. ( D ) Histogram shows distribution of H3K4me2 lncRNAs with antisense (X) and sense (S) pattern with respect to their protein coding partner (pPCGs). ‘n’ denotes number of lncRNA-pPCG pairs in each pattern. ( E ) Genomic organization of H3K4me2 lncRNAs (green bars) with respect to nearest protein coding genes (grey bars). XH: where lncRNA shows Head-to-Head arrangement with nearby protein coding gene. XT: lncRNA in Tail-to-Tail arrangement with protein coding gene, XI: lncRNA located inside a protein coding gene, XO: lncRNA located outside and covers the entire protein coding gene. Sense pairs means lncRNAs in the same orientation as protein coding gene. The notion of greater or less than 2 kb means that the partner genes (pPCGs) are located within 2 kb (<2 kb) or away from 2 kb (> 2 kb) but within 50 kb window with respect to H3K4me2 lncRNAs. ( F ) Distribution of different patterns of genomic arrangements as described in (E) for H3K4me2 lncRNAs and non-CAR lncRNAs.
    Figure Legend Snippet: Characterizing H3K4me2 enriched lncRNAs. ( A ) ChRIP experimental workflow to identify lncRNAs bound to chromatin enriched with H3K4me2 and WDR5 in BT-549 cell line. ( B ) Computational approach used for finding chromatin-associated RNAs and enriched patterns of genomic organization with respect to nearest protein coding genes. ( C ) Scatter plot showing the enrichment of H3K4me2 lncRNAs over input. X-axis denotes log transformed expression values of H3K4me2 lncRNAs and Y-axis denotes log-fold changes of lncRNAs in H3K4me2 ChRIP sample over nuclear input sample. ( D ) Histogram shows distribution of H3K4me2 lncRNAs with antisense (X) and sense (S) pattern with respect to their protein coding partner (pPCGs). ‘n’ denotes number of lncRNA-pPCG pairs in each pattern. ( E ) Genomic organization of H3K4me2 lncRNAs (green bars) with respect to nearest protein coding genes (grey bars). XH: where lncRNA shows Head-to-Head arrangement with nearby protein coding gene. XT: lncRNA in Tail-to-Tail arrangement with protein coding gene, XI: lncRNA located inside a protein coding gene, XO: lncRNA located outside and covers the entire protein coding gene. Sense pairs means lncRNAs in the same orientation as protein coding gene. The notion of greater or less than 2 kb means that the partner genes (pPCGs) are located within 2 kb (<2 kb) or away from 2 kb (> 2 kb) but within 50 kb window with respect to H3K4me2 lncRNAs. ( F ) Distribution of different patterns of genomic arrangements as described in (E) for H3K4me2 lncRNAs and non-CAR lncRNAs.

    Techniques Used: Transformation Assay, Expressing

    H3K4me2 XH lncRNAs regulate the transcriptional activity of their protein coding partners. ( A ) Nuclear expression levels of H3K4me2 XH lncRNAs and non-CAR XH lncRNAs. ( B ) Expression of H3K4me2 XH lncRNA-protein coding pairs (pPCGs) in nuclear input and H3K4me2 ChRIP samples. The P -value denotes the significance of difference between nuclear input and H3K4me2. ( C ) Gene ontology based functional enrichment analysis of protein coding genes that are partners (pPCGs) to H3K4me2 lncRNAs: divergent (XH), other antisense (XT, XI, and XO) and sense lncRNAs. The bar graph shows number of genes in corresponding ontology term and the heatmap represents the significance of individual terms ( P -values obtained using GeneSCF). The highlighted box, by the heatmap, is listed with different transcription factors from the enriched transcription related terms. ( D ) Gene expression analysis of three transcription factors ( FOXD3, GATA6 and HOXC13 ) using RT–qPCR analysis following downregulation of their neighboring H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) with siRNA in BT-549 cells. LncRNA expression is in green bars while transcription factors expression is in grey. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( E ) RT–qPCR analysis of FOXD3, GATA6 and HOXC13 gene expression after strand-specific downregulation of FOXD3-AS1, GATA6-AS1 and HOXC13-AS respectively, using LNA. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( F ) Gene expression analysis of three H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) using RT–qPCR analysis following downregulation of their respective transcription factor partners with siRNA or esiRNA in BT-549 cells. LncRNAs expression is depicted as green bars while transcription factors expression is in grey bars. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ns denotes non-significant.
    Figure Legend Snippet: H3K4me2 XH lncRNAs regulate the transcriptional activity of their protein coding partners. ( A ) Nuclear expression levels of H3K4me2 XH lncRNAs and non-CAR XH lncRNAs. ( B ) Expression of H3K4me2 XH lncRNA-protein coding pairs (pPCGs) in nuclear input and H3K4me2 ChRIP samples. The P -value denotes the significance of difference between nuclear input and H3K4me2. ( C ) Gene ontology based functional enrichment analysis of protein coding genes that are partners (pPCGs) to H3K4me2 lncRNAs: divergent (XH), other antisense (XT, XI, and XO) and sense lncRNAs. The bar graph shows number of genes in corresponding ontology term and the heatmap represents the significance of individual terms ( P -values obtained using GeneSCF). The highlighted box, by the heatmap, is listed with different transcription factors from the enriched transcription related terms. ( D ) Gene expression analysis of three transcription factors ( FOXD3, GATA6 and HOXC13 ) using RT–qPCR analysis following downregulation of their neighboring H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) with siRNA in BT-549 cells. LncRNA expression is in green bars while transcription factors expression is in grey. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( E ) RT–qPCR analysis of FOXD3, GATA6 and HOXC13 gene expression after strand-specific downregulation of FOXD3-AS1, GATA6-AS1 and HOXC13-AS respectively, using LNA. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( F ) Gene expression analysis of three H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) using RT–qPCR analysis following downregulation of their respective transcription factor partners with siRNA or esiRNA in BT-549 cells. LncRNAs expression is depicted as green bars while transcription factors expression is in grey bars. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ns denotes non-significant.

    Techniques Used: Activity Assay, Expressing, Functional Assay, Quantitative RT-PCR, esiRNA

    XH lncRNAs are overrepresented in active lncCARs. ( A ) Scatter plot showing enrichment of WDR5 lncRNAs over nuclear input. The X-axis denotes log transformed expression and Y-axis denotes log-fold change of WDR5 lncRNAs over nuclear input. Venn diagram, in the Scatter plot, shows 209 lncRNAs that are commonly enriched in both H3K4me2 and WDR5 ChRIP pulldowns. ( B ) Distribution of different patterns of genomic arrangements as described in Figure for WDR5 lncRNAs and non-CAR lncRNAs. ( C ) Distribution of different patterns of genomic arrangements as described in Figure for active lncRNA (ChRIP pulldown using H3K4me2 and WDR5 antibodies) and inactive lncRNA (ChRIP pulldown using EZH2 and H3K27me3 antibodies). ( D ) Nuclear expression levels of WDR5 XH lncRNAs, active XH lncCAR (WDR5 and H3K4me2) and non-CAR XH lncRNAs. ( E ) Enrichment of H3K4me2, H3K4me3, WDR5 ChIP-seq signals over active XH lncCAR (in green, n = 98) and non-CAR XH lncRNA (in black, n = 335) promoters (±2 kb) in BT-549 cells. The signals presented in the plots represent log 2 ratio between ChIP and input sample. TSS denotes the transcription start site of lncRNAs. ( F ) RT-qPCR analysis of WDR5 UV-RIP. The Y-axis shows fold enrichment relative to IgG. The significance of FOXD3-AS1 and HOXC13-AS enrichment in WDR5 RIP is calculated compared to the enrichment of FOXD3 and HOXC13 mRNAs, respectively. *** P ≤ 0.001. ( G ) Left panel: f-RIP of WDR5 WT and WDR5 F266A using FLAG antibody. The Y-axis shows percentage of Input. The significance of decrease in FOXD3-AS1, HOXC13-AS and positive control HOTTIP enrichment in WDR5 F266A RIP is calculated compared to their respective enrichments in WDR5-WT. GAPDH was used as negative control. Right panel: Expression of FLAG tagged WDR5 WT and WDR5 F266A mutant in HeLa cells by western blot. Data are shown as mean ± SD. ** P ≤ 0.01.
    Figure Legend Snippet: XH lncRNAs are overrepresented in active lncCARs. ( A ) Scatter plot showing enrichment of WDR5 lncRNAs over nuclear input. The X-axis denotes log transformed expression and Y-axis denotes log-fold change of WDR5 lncRNAs over nuclear input. Venn diagram, in the Scatter plot, shows 209 lncRNAs that are commonly enriched in both H3K4me2 and WDR5 ChRIP pulldowns. ( B ) Distribution of different patterns of genomic arrangements as described in Figure for WDR5 lncRNAs and non-CAR lncRNAs. ( C ) Distribution of different patterns of genomic arrangements as described in Figure for active lncRNA (ChRIP pulldown using H3K4me2 and WDR5 antibodies) and inactive lncRNA (ChRIP pulldown using EZH2 and H3K27me3 antibodies). ( D ) Nuclear expression levels of WDR5 XH lncRNAs, active XH lncCAR (WDR5 and H3K4me2) and non-CAR XH lncRNAs. ( E ) Enrichment of H3K4me2, H3K4me3, WDR5 ChIP-seq signals over active XH lncCAR (in green, n = 98) and non-CAR XH lncRNA (in black, n = 335) promoters (±2 kb) in BT-549 cells. The signals presented in the plots represent log 2 ratio between ChIP and input sample. TSS denotes the transcription start site of lncRNAs. ( F ) RT-qPCR analysis of WDR5 UV-RIP. The Y-axis shows fold enrichment relative to IgG. The significance of FOXD3-AS1 and HOXC13-AS enrichment in WDR5 RIP is calculated compared to the enrichment of FOXD3 and HOXC13 mRNAs, respectively. *** P ≤ 0.001. ( G ) Left panel: f-RIP of WDR5 WT and WDR5 F266A using FLAG antibody. The Y-axis shows percentage of Input. The significance of decrease in FOXD3-AS1, HOXC13-AS and positive control HOTTIP enrichment in WDR5 F266A RIP is calculated compared to their respective enrichments in WDR5-WT. GAPDH was used as negative control. Right panel: Expression of FLAG tagged WDR5 WT and WDR5 F266A mutant in HeLa cells by western blot. Data are shown as mean ± SD. ** P ≤ 0.01.

    Techniques Used: Transformation Assay, Expressing, ChIP-sequencing, Quantitative RT-PCR, Positive Control, Negative Control, Mutagenesis, Western Blot

    Active XH lncCARs promote transcription of their protein coding partners. ( A and B ) Promoter targeting of active XH lncCARs detected by ChOP. A) qPCR analysis of ChOP pull-downs, performed using FOXD3-AS1 and HOXC13-AS antisense probes, with primers over TSS (transcription start sites), regions spanning upstream and downstream of TSS and over transcription termination sites (TTS) of FOXD3 and HOXC13 genes in BT-549 cells. The ChOP pull-down with LacZ antisense oligo, used as a negative control (grey bars). Specific enrichment pattern for each of the XH lncCARs is depicted in green bars. The schematic in the right of the bar graph represents the genomic locations of the respective primers (grey: not enriched and rosetta: showing enrichment). Data are shown as mean ± SD (n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( B ) Similar qPCR analysis of ChOP pull-downs using FOXD3-AS1 and HOXC13-AS antisense probes upon ActD treatment in BT-549 cells. Data are shown as mean ± SD ( n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( C ) RT-qPCR analysis to detect relative levels of nascent FOXD3 and HOXC13 transcripts by Click-iT at 48 hours after the knockdown of FOXD3-AS1 and HOXC13-AS . Bar graph depicts the level of nascent protein coding transcripts upon removal of their respective active XH lncCARs. Data represent the mean ± SD of two independent biological experiments. * if P ≤ 0.05 and ** if P ≤ 0.01. ( D and E ) ChIP-qPCR analysis of the enrichment of H3K4me2 and H3K4me3 at the promoter region of FOXD3 (D) and HOXC13 (E) upon down regulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of H3K4me2 and H3K4me3 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA.The locations of ChIP primers used are depicted in schematic in the bottm of each bar graph. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( F and G ) ChIP-qPCR analysis of enrichment of WDR5 at the promoter region of FOXD3(F) and HOXC13 (G) upon downregulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of WDR5 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA. The ChIP primers used in the experiment are same as described in panel D–E. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( H and I ) FOXD3-AS1 and HOXC13-AS overexpression: RT–qPCR analysis of FOXD3 expression, upon ectopic overexpression of FOXD3-AS1 (H) and HOXC13-AS transcript (I) respectively, at Day 2 and Day 5 post transfection in BT-549 cell line. pcDNA empty vector transfection used as a control. Data represent the mean ± SD. of two independent biological experiments. P -values has been denoted as * if P ≤ 0.05, ** if P ≤ 0.01. ns denotes non-significant. ( J ) Cell fractionation in BT-549 cells show distribution of the overexpressed FOXD3-AS1 and HOXC13-AS transcripts. Data represent the mean ± SD of two independent biological experiments. GAPDH serves as positive control for cytoplasmic fraction, U6 and KCNQ1OT1 serves as positive control for nuclear fraction.
    Figure Legend Snippet: Active XH lncCARs promote transcription of their protein coding partners. ( A and B ) Promoter targeting of active XH lncCARs detected by ChOP. A) qPCR analysis of ChOP pull-downs, performed using FOXD3-AS1 and HOXC13-AS antisense probes, with primers over TSS (transcription start sites), regions spanning upstream and downstream of TSS and over transcription termination sites (TTS) of FOXD3 and HOXC13 genes in BT-549 cells. The ChOP pull-down with LacZ antisense oligo, used as a negative control (grey bars). Specific enrichment pattern for each of the XH lncCARs is depicted in green bars. The schematic in the right of the bar graph represents the genomic locations of the respective primers (grey: not enriched and rosetta: showing enrichment). Data are shown as mean ± SD (n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( B ) Similar qPCR analysis of ChOP pull-downs using FOXD3-AS1 and HOXC13-AS antisense probes upon ActD treatment in BT-549 cells. Data are shown as mean ± SD ( n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( C ) RT-qPCR analysis to detect relative levels of nascent FOXD3 and HOXC13 transcripts by Click-iT at 48 hours after the knockdown of FOXD3-AS1 and HOXC13-AS . Bar graph depicts the level of nascent protein coding transcripts upon removal of their respective active XH lncCARs. Data represent the mean ± SD of two independent biological experiments. * if P ≤ 0.05 and ** if P ≤ 0.01. ( D and E ) ChIP-qPCR analysis of the enrichment of H3K4me2 and H3K4me3 at the promoter region of FOXD3 (D) and HOXC13 (E) upon down regulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of H3K4me2 and H3K4me3 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA.The locations of ChIP primers used are depicted in schematic in the bottm of each bar graph. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( F and G ) ChIP-qPCR analysis of enrichment of WDR5 at the promoter region of FOXD3(F) and HOXC13 (G) upon downregulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of WDR5 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA. The ChIP primers used in the experiment are same as described in panel D–E. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( H and I ) FOXD3-AS1 and HOXC13-AS overexpression: RT–qPCR analysis of FOXD3 expression, upon ectopic overexpression of FOXD3-AS1 (H) and HOXC13-AS transcript (I) respectively, at Day 2 and Day 5 post transfection in BT-549 cell line. pcDNA empty vector transfection used as a control. Data represent the mean ± SD. of two independent biological experiments. P -values has been denoted as * if P ≤ 0.05, ** if P ≤ 0.01. ns denotes non-significant. ( J ) Cell fractionation in BT-549 cells show distribution of the overexpressed FOXD3-AS1 and HOXC13-AS transcripts. Data represent the mean ± SD of two independent biological experiments. GAPDH serves as positive control for cytoplasmic fraction, U6 and KCNQ1OT1 serves as positive control for nuclear fraction.

    Techniques Used: Negative Control, Quantitative RT-PCR, IF-P, Over Expression, Expressing, Transfection, Plasmid Preparation, Cell Fractionation, Positive Control

    bt 549 cells  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH bt 549 cells
    Localization of FHOD1 and INF2 in basal-like breast cancer cell lines. (A) FHOD1 (red) is mostly seen as cytoplasmic dots in <t>BT-549</t> and MDA-MB-231 cells. Clear co-localization with actin filament bundles (green) can be seen. (B) INF2 (red) is also distributed as dots in the cytoplasm. Co-localization with actin filaments (green) is present in both cell lines. Right panel shows higher magnification images of the marked (white box) areas.
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    1) Product Images from "Formin Proteins FHOD1 and INF2 in Triple-Negative Breast Cancer: Association With Basal Markers and Functional Activities"

    Article Title: Formin Proteins FHOD1 and INF2 in Triple-Negative Breast Cancer: Association With Basal Markers and Functional Activities

    Journal: Breast Cancer : Basic and Clinical Research

    doi: 10.1177/1178223418792247

    Localization of FHOD1 and INF2 in basal-like breast cancer cell lines. (A) FHOD1 (red) is mostly seen as cytoplasmic dots in BT-549 and MDA-MB-231 cells. Clear co-localization with actin filament bundles (green) can be seen. (B) INF2 (red) is also distributed as dots in the cytoplasm. Co-localization with actin filaments (green) is present in both cell lines. Right panel shows higher magnification images of the marked (white box) areas.
    Figure Legend Snippet: Localization of FHOD1 and INF2 in basal-like breast cancer cell lines. (A) FHOD1 (red) is mostly seen as cytoplasmic dots in BT-549 and MDA-MB-231 cells. Clear co-localization with actin filament bundles (green) can be seen. (B) INF2 (red) is also distributed as dots in the cytoplasm. Co-localization with actin filaments (green) is present in both cell lines. Right panel shows higher magnification images of the marked (white box) areas.

    Techniques Used:

    Knockdown of FHOD1 and INF2 expression is accompanied by morphological and functional alterations in basal-like breast cancer cell lines. (A) Western blotting confirms that the expression of both formins is markedly reduced by siRNA treatment. (B) Quantification of Transwell migration experiments show that migration is significantly reduced in both cell lines on knockdown of FHOD1 or INF2. (C) The cell area significantly increases on FHOD1 or INF2 knockdown in BT-549 cells as compared with control cells. Cellular axis ratio is decreased, indicating that cells were less elongated and more round than control cells. The reduction of axis ratio is statistically significant only for INF2 knockdown. (D) Knockdown of INF2 alters the morphology of MDA-MB-231 cells in a similar way: cell area and roundness are increased. FHOD1 knockdown has a minor effect on morphology in this cell line. (E) Graph illustrating wound confluence of BT-549 cells as a function of time. Wound healing and invasion is significantly slower after FHOD1 and INF2 depletion. (F) Graphs from wound healing and invasion assays using MDA-MB-231 cells. Wound healing and invasion is slower in FHOD1 or INF2 knockdown groups than control cells. (G) The proliferation index, measured as percent of Ki-67 positive cells, is reduced by FHOD1 and especially by INF2 depletion. Error bars indicate standard deviation. AR indicates axis ratio; OD, standardised extracted crystal violet optical density; px, pixels. * P ⩽ .05; ** P ⩽ .01; *** P ⩽ .001.
    Figure Legend Snippet: Knockdown of FHOD1 and INF2 expression is accompanied by morphological and functional alterations in basal-like breast cancer cell lines. (A) Western blotting confirms that the expression of both formins is markedly reduced by siRNA treatment. (B) Quantification of Transwell migration experiments show that migration is significantly reduced in both cell lines on knockdown of FHOD1 or INF2. (C) The cell area significantly increases on FHOD1 or INF2 knockdown in BT-549 cells as compared with control cells. Cellular axis ratio is decreased, indicating that cells were less elongated and more round than control cells. The reduction of axis ratio is statistically significant only for INF2 knockdown. (D) Knockdown of INF2 alters the morphology of MDA-MB-231 cells in a similar way: cell area and roundness are increased. FHOD1 knockdown has a minor effect on morphology in this cell line. (E) Graph illustrating wound confluence of BT-549 cells as a function of time. Wound healing and invasion is significantly slower after FHOD1 and INF2 depletion. (F) Graphs from wound healing and invasion assays using MDA-MB-231 cells. Wound healing and invasion is slower in FHOD1 or INF2 knockdown groups than control cells. (G) The proliferation index, measured as percent of Ki-67 positive cells, is reduced by FHOD1 and especially by INF2 depletion. Error bars indicate standard deviation. AR indicates axis ratio; OD, standardised extracted crystal violet optical density; px, pixels. * P ⩽ .05; ** P ⩽ .01; *** P ⩽ .001.

    Techniques Used: Expressing, Functional Assay, Western Blot, Migration, Standard Deviation

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    CLS Cell Lines Service GmbH bt 549
    ( A ) TNBC and ( B ) non-TNBC cells were incubated for 24 h in medium with or without glutamine (2 mM) before incubation in the presence or absence of TRAIL for 24 h (100 ng/ml MDA-MB468, 50 ng/ml MDA-MB231, 10 ng/ml MDA-MB436, 100 ng/ml <t>BT549,</t> 500 ng/ml non-TNBC). Apoptosis was assessed as described in Material and Methods section. Error bars represent s.d. from three independent experiments. ** P < 0.01, *** P < 0.001. ( C ) Cells were incubated in medium with or without glutamine as described in ( A ) before treating them with TRAIL for the times indicated. Caspase-8 activation was examined by western blotting. GAPDH was used as a protein loading control. Results shown are representative of at least three different experiments.
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    1) Product Images from "Glutamine metabolism regulates FLIP expression and sensitivity to TRAIL in triple-negative breast cancer cells"

    Article Title: Glutamine metabolism regulates FLIP expression and sensitivity to TRAIL in triple-negative breast cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0263-0

    ( A ) TNBC and ( B ) non-TNBC cells were incubated for 24 h in medium with or without glutamine (2 mM) before incubation in the presence or absence of TRAIL for 24 h (100 ng/ml MDA-MB468, 50 ng/ml MDA-MB231, 10 ng/ml MDA-MB436, 100 ng/ml BT549, 500 ng/ml non-TNBC). Apoptosis was assessed as described in Material and Methods section. Error bars represent s.d. from three independent experiments. ** P < 0.01, *** P < 0.001. ( C ) Cells were incubated in medium with or without glutamine as described in ( A ) before treating them with TRAIL for the times indicated. Caspase-8 activation was examined by western blotting. GAPDH was used as a protein loading control. Results shown are representative of at least three different experiments.
    Figure Legend Snippet: ( A ) TNBC and ( B ) non-TNBC cells were incubated for 24 h in medium with or without glutamine (2 mM) before incubation in the presence or absence of TRAIL for 24 h (100 ng/ml MDA-MB468, 50 ng/ml MDA-MB231, 10 ng/ml MDA-MB436, 100 ng/ml BT549, 500 ng/ml non-TNBC). Apoptosis was assessed as described in Material and Methods section. Error bars represent s.d. from three independent experiments. ** P < 0.01, *** P < 0.001. ( C ) Cells were incubated in medium with or without glutamine as described in ( A ) before treating them with TRAIL for the times indicated. Caspase-8 activation was examined by western blotting. GAPDH was used as a protein loading control. Results shown are representative of at least three different experiments.

    Techniques Used: Incubation, Activation Assay, Western Blot

    ( A ) MDA-MB468 cells were incubated for 24 h in medium with or without glutamine and the indicated additions prior to treatment in the presence or absence of TRAIL (100 ng/ml) for 24 h. Apoptosis was measured as described in Materials and Methods section. ( B ) TNBC cells incubated in glutamine-containing medium with or without AOA for 24 h were treated in the presence or absence of TRAIL for 24 h (100 ng/ml MDA-MB468, 50 ng/ml MDA-MB231, 10 ng/ml MDA-MB436, 100 ng/ml BT549) and apoptosis was then determined. ( C ) Apoptosis in MDA-MB468 cells incubated in medium with glutamine and AOA in the presence or absence of non-essential amino acids (NEAA) before treatment with or without TRAIL (100 ng/ml). ( D ) MDA-MB468 cells were treated as indicated and TRAIL-R2 levels were assessed by western blotting or flow-cytometry. FLIP levels were determined by western blotting. Results shown are representative of three independent experiments. ( E ) Protein synthesis in MDA-MB468 cells incubated in glutamine-containing medium and the metabolic inhibitor AOA in presence or absence of NEAA for 6 h (left panel). (Right panel) MDA-MB468 cells were incubated as indicated for 24 h and FLIP levels were assessed by western blotting. GAPDH was used as a protein loading control. In panels A , B , C and E , error bars represent s.d. from three independent experiments. ** P < 0.01, *** P < 0.001, n.s. not statistically significant.
    Figure Legend Snippet: ( A ) MDA-MB468 cells were incubated for 24 h in medium with or without glutamine and the indicated additions prior to treatment in the presence or absence of TRAIL (100 ng/ml) for 24 h. Apoptosis was measured as described in Materials and Methods section. ( B ) TNBC cells incubated in glutamine-containing medium with or without AOA for 24 h were treated in the presence or absence of TRAIL for 24 h (100 ng/ml MDA-MB468, 50 ng/ml MDA-MB231, 10 ng/ml MDA-MB436, 100 ng/ml BT549) and apoptosis was then determined. ( C ) Apoptosis in MDA-MB468 cells incubated in medium with glutamine and AOA in the presence or absence of non-essential amino acids (NEAA) before treatment with or without TRAIL (100 ng/ml). ( D ) MDA-MB468 cells were treated as indicated and TRAIL-R2 levels were assessed by western blotting or flow-cytometry. FLIP levels were determined by western blotting. Results shown are representative of three independent experiments. ( E ) Protein synthesis in MDA-MB468 cells incubated in glutamine-containing medium and the metabolic inhibitor AOA in presence or absence of NEAA for 6 h (left panel). (Right panel) MDA-MB468 cells were incubated as indicated for 24 h and FLIP levels were assessed by western blotting. GAPDH was used as a protein loading control. In panels A , B , C and E , error bars represent s.d. from three independent experiments. ** P < 0.01, *** P < 0.001, n.s. not statistically significant.

    Techniques Used: Incubation, Western Blot, Flow Cytometry

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    CLS Cell Lines Service GmbH bt 549 cells
    a , b Immunofluorescence images of A549 cells ( a ) and <t>BT-549</t> ( b ) showing dsRNA-specific J2 signals in hnRNPK KD cells. The panel to the right shows the merged images. Magnified J2 signals (puncta) are shown in an inset at the upper right corner indicated by white box. Indicative scale bar is 50um. Graphs show the number of J2 signals in hnRNPK KD cells quantified using Imaris spot detection tools. For A549 cell line - control: n = 59, siRNA1: n = 64 and siRNA2: n = 73 cells and for BT-549: control: n = 25, siRNA1: n = 37 and siRNA2: n = 44 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. Significance was calculated by One-way ANOVA (ordinary multiple comparisons test). c , e RNAscope images of A549 cells ( c ) and BT-549 cells ( e ) showing IER3 (red) and IER3-AS1 (green) in control and hnRNPK KD cells. Panel to the right shows the magnified images of the merged panels indicated by white box. White arrows marks IER3-AS1 (green dots) and yellow arrows marks colocalized IER3/IER3-AS1 dsRNA (Yellow dots). Indicative scale bar on the images is 50 um. Bar graph shows the number of IER3-AS1 and IER3 RNA signals per cell in control and hnRNPK KD cells quantified using Imaris spot detection tools. For A549 - control: n = 101, siRNA1: n = 78 and siRNA2: n = 97 cells and for BT-549 - control: n = 132, siRNA1: n = 44 and siRNA2: n = F36 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. The significance was calculated by Two-way ANOVA (Sidak’s multiple comparisons test). d Immunoblot showing the levels of HnRNPK and IER3 in hnRNPK KD A549 cells. GAPDH was used as an internal loading control. Similar results were observed in an independent biological replicate. Histograms showing the relative band intensities from the western blot are shown left side. The p values were calculated using two-sided Student’s t -test and data are presented ± SD from two independent replicates.
    Bt 549 Cells, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CLS Cell Lines Service GmbH bt549
    a , b Immunofluorescence images of A549 cells ( a ) and <t>BT-549</t> ( b ) showing dsRNA-specific J2 signals in hnRNPK KD cells. The panel to the right shows the merged images. Magnified J2 signals (puncta) are shown in an inset at the upper right corner indicated by white box. Indicative scale bar is 50um. Graphs show the number of J2 signals in hnRNPK KD cells quantified using Imaris spot detection tools. For A549 cell line - control: n = 59, siRNA1: n = 64 and siRNA2: n = 73 cells and for BT-549: control: n = 25, siRNA1: n = 37 and siRNA2: n = 44 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. Significance was calculated by One-way ANOVA (ordinary multiple comparisons test). c , e RNAscope images of A549 cells ( c ) and BT-549 cells ( e ) showing IER3 (red) and IER3-AS1 (green) in control and hnRNPK KD cells. Panel to the right shows the magnified images of the merged panels indicated by white box. White arrows marks IER3-AS1 (green dots) and yellow arrows marks colocalized IER3/IER3-AS1 dsRNA (Yellow dots). Indicative scale bar on the images is 50 um. Bar graph shows the number of IER3-AS1 and IER3 RNA signals per cell in control and hnRNPK KD cells quantified using Imaris spot detection tools. For A549 - control: n = 101, siRNA1: n = 78 and siRNA2: n = 97 cells and for BT-549 - control: n = 132, siRNA1: n = 44 and siRNA2: n = F36 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. The significance was calculated by Two-way ANOVA (Sidak’s multiple comparisons test). d Immunoblot showing the levels of HnRNPK and IER3 in hnRNPK KD A549 cells. GAPDH was used as an internal loading control. Similar results were observed in an independent biological replicate. Histograms showing the relative band intensities from the western blot are shown left side. The p values were calculated using two-sided Student’s t -test and data are presented ± SD from two independent replicates.
    Bt549, supplied by CLS Cell Lines Service GmbH, 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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    CLS Cell Lines Service GmbH humantnbc cells
    a , b Immunofluorescence images of A549 cells ( a ) and <t>BT-549</t> ( b ) showing dsRNA-specific J2 signals in hnRNPK KD cells. The panel to the right shows the merged images. Magnified J2 signals (puncta) are shown in an inset at the upper right corner indicated by white box. Indicative scale bar is 50um. Graphs show the number of J2 signals in hnRNPK KD cells quantified using Imaris spot detection tools. For A549 cell line - control: n = 59, siRNA1: n = 64 and siRNA2: n = 73 cells and for BT-549: control: n = 25, siRNA1: n = 37 and siRNA2: n = 44 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. Significance was calculated by One-way ANOVA (ordinary multiple comparisons test). c , e RNAscope images of A549 cells ( c ) and BT-549 cells ( e ) showing IER3 (red) and IER3-AS1 (green) in control and hnRNPK KD cells. Panel to the right shows the magnified images of the merged panels indicated by white box. White arrows marks IER3-AS1 (green dots) and yellow arrows marks colocalized IER3/IER3-AS1 dsRNA (Yellow dots). Indicative scale bar on the images is 50 um. Bar graph shows the number of IER3-AS1 and IER3 RNA signals per cell in control and hnRNPK KD cells quantified using Imaris spot detection tools. For A549 - control: n = 101, siRNA1: n = 78 and siRNA2: n = 97 cells and for BT-549 - control: n = 132, siRNA1: n = 44 and siRNA2: n = F36 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. The significance was calculated by Two-way ANOVA (Sidak’s multiple comparisons test). d Immunoblot showing the levels of HnRNPK and IER3 in hnRNPK KD A549 cells. GAPDH was used as an internal loading control. Similar results were observed in an independent biological replicate. Histograms showing the relative band intensities from the western blot are shown left side. The p values were calculated using two-sided Student’s t -test and data are presented ± SD from two independent replicates.
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    CLS Cell Lines Service GmbH bt 549
    Characterizing H3K4me2 enriched lncRNAs. ( A ) ChRIP experimental workflow to identify lncRNAs bound to chromatin enriched with H3K4me2 and WDR5 in <t>BT-549</t> cell line. ( B ) Computational approach used for finding chromatin-associated RNAs and enriched patterns of genomic organization with respect to nearest protein coding genes. ( C ) Scatter plot showing the enrichment of H3K4me2 lncRNAs over input. X-axis denotes log transformed expression values of H3K4me2 lncRNAs and Y-axis denotes log-fold changes of lncRNAs in H3K4me2 ChRIP sample over nuclear input sample. ( D ) Histogram shows distribution of H3K4me2 lncRNAs with antisense (X) and sense (S) pattern with respect to their protein coding partner (pPCGs). ‘n’ denotes number of lncRNA-pPCG pairs in each pattern. ( E ) Genomic organization of H3K4me2 lncRNAs (green bars) with respect to nearest protein coding genes (grey bars). XH: where lncRNA shows Head-to-Head arrangement with nearby protein coding gene. XT: lncRNA in Tail-to-Tail arrangement with protein coding gene, XI: lncRNA located inside a protein coding gene, XO: lncRNA located outside and covers the entire protein coding gene. Sense pairs means lncRNAs in the same orientation as protein coding gene. The notion of greater or less than 2 kb means that the partner genes (pPCGs) are located within 2 kb (<2 kb) or away from 2 kb (> 2 kb) but within 50 kb window with respect to H3K4me2 lncRNAs. ( F ) Distribution of different patterns of genomic arrangements as described in (E) for H3K4me2 lncRNAs and non-CAR lncRNAs.
    Bt 549, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    a , b Immunofluorescence images of A549 cells ( a ) and BT-549 ( b ) showing dsRNA-specific J2 signals in hnRNPK KD cells. The panel to the right shows the merged images. Magnified J2 signals (puncta) are shown in an inset at the upper right corner indicated by white box. Indicative scale bar is 50um. Graphs show the number of J2 signals in hnRNPK KD cells quantified using Imaris spot detection tools. For A549 cell line - control: n = 59, siRNA1: n = 64 and siRNA2: n = 73 cells and for BT-549: control: n = 25, siRNA1: n = 37 and siRNA2: n = 44 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. Significance was calculated by One-way ANOVA (ordinary multiple comparisons test). c , e RNAscope images of A549 cells ( c ) and BT-549 cells ( e ) showing IER3 (red) and IER3-AS1 (green) in control and hnRNPK KD cells. Panel to the right shows the magnified images of the merged panels indicated by white box. White arrows marks IER3-AS1 (green dots) and yellow arrows marks colocalized IER3/IER3-AS1 dsRNA (Yellow dots). Indicative scale bar on the images is 50 um. Bar graph shows the number of IER3-AS1 and IER3 RNA signals per cell in control and hnRNPK KD cells quantified using Imaris spot detection tools. For A549 - control: n = 101, siRNA1: n = 78 and siRNA2: n = 97 cells and for BT-549 - control: n = 132, siRNA1: n = 44 and siRNA2: n = F36 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. The significance was calculated by Two-way ANOVA (Sidak’s multiple comparisons test). d Immunoblot showing the levels of HnRNPK and IER3 in hnRNPK KD A549 cells. GAPDH was used as an internal loading control. Similar results were observed in an independent biological replicate. Histograms showing the relative band intensities from the western blot are shown left side. The p values were calculated using two-sided Student’s t -test and data are presented ± SD from two independent replicates.

    Journal: Nature Communications

    Article Title: HnRNPK maintains single strand RNA through controlling double-strand RNA in mammalian cells

    doi: 10.1038/s41467-022-32537-0

    Figure Lengend Snippet: a , b Immunofluorescence images of A549 cells ( a ) and BT-549 ( b ) showing dsRNA-specific J2 signals in hnRNPK KD cells. The panel to the right shows the merged images. Magnified J2 signals (puncta) are shown in an inset at the upper right corner indicated by white box. Indicative scale bar is 50um. Graphs show the number of J2 signals in hnRNPK KD cells quantified using Imaris spot detection tools. For A549 cell line - control: n = 59, siRNA1: n = 64 and siRNA2: n = 73 cells and for BT-549: control: n = 25, siRNA1: n = 37 and siRNA2: n = 44 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. Significance was calculated by One-way ANOVA (ordinary multiple comparisons test). c , e RNAscope images of A549 cells ( c ) and BT-549 cells ( e ) showing IER3 (red) and IER3-AS1 (green) in control and hnRNPK KD cells. Panel to the right shows the magnified images of the merged panels indicated by white box. White arrows marks IER3-AS1 (green dots) and yellow arrows marks colocalized IER3/IER3-AS1 dsRNA (Yellow dots). Indicative scale bar on the images is 50 um. Bar graph shows the number of IER3-AS1 and IER3 RNA signals per cell in control and hnRNPK KD cells quantified using Imaris spot detection tools. For A549 - control: n = 101, siRNA1: n = 78 and siRNA2: n = 97 cells and for BT-549 - control: n = 132, siRNA1: n = 44 and siRNA2: n = F36 cells were counted from three independent experiments. Data are presented as mean values +/−SEM. The significance was calculated by Two-way ANOVA (Sidak’s multiple comparisons test). d Immunoblot showing the levels of HnRNPK and IER3 in hnRNPK KD A549 cells. GAPDH was used as an internal loading control. Similar results were observed in an independent biological replicate. Histograms showing the relative band intensities from the western blot are shown left side. The p values were calculated using two-sided Student’s t -test and data are presented ± SD from two independent replicates.

    Article Snippet: HEK293T, HeLa, A549 and BT-549 cells were obtained from CLS Cell Lines Service.

    Techniques: Immunofluorescence, Western Blot

    Characterizing H3K4me2 enriched lncRNAs. ( A ) ChRIP experimental workflow to identify lncRNAs bound to chromatin enriched with H3K4me2 and WDR5 in BT-549 cell line. ( B ) Computational approach used for finding chromatin-associated RNAs and enriched patterns of genomic organization with respect to nearest protein coding genes. ( C ) Scatter plot showing the enrichment of H3K4me2 lncRNAs over input. X-axis denotes log transformed expression values of H3K4me2 lncRNAs and Y-axis denotes log-fold changes of lncRNAs in H3K4me2 ChRIP sample over nuclear input sample. ( D ) Histogram shows distribution of H3K4me2 lncRNAs with antisense (X) and sense (S) pattern with respect to their protein coding partner (pPCGs). ‘n’ denotes number of lncRNA-pPCG pairs in each pattern. ( E ) Genomic organization of H3K4me2 lncRNAs (green bars) with respect to nearest protein coding genes (grey bars). XH: where lncRNA shows Head-to-Head arrangement with nearby protein coding gene. XT: lncRNA in Tail-to-Tail arrangement with protein coding gene, XI: lncRNA located inside a protein coding gene, XO: lncRNA located outside and covers the entire protein coding gene. Sense pairs means lncRNAs in the same orientation as protein coding gene. The notion of greater or less than 2 kb means that the partner genes (pPCGs) are located within 2 kb (<2 kb) or away from 2 kb (> 2 kb) but within 50 kb window with respect to H3K4me2 lncRNAs. ( F ) Distribution of different patterns of genomic arrangements as described in (E) for H3K4me2 lncRNAs and non-CAR lncRNAs.

    Journal: Nucleic Acids Research

    Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

    doi: 10.1093/nar/gky635

    Figure Lengend Snippet: Characterizing H3K4me2 enriched lncRNAs. ( A ) ChRIP experimental workflow to identify lncRNAs bound to chromatin enriched with H3K4me2 and WDR5 in BT-549 cell line. ( B ) Computational approach used for finding chromatin-associated RNAs and enriched patterns of genomic organization with respect to nearest protein coding genes. ( C ) Scatter plot showing the enrichment of H3K4me2 lncRNAs over input. X-axis denotes log transformed expression values of H3K4me2 lncRNAs and Y-axis denotes log-fold changes of lncRNAs in H3K4me2 ChRIP sample over nuclear input sample. ( D ) Histogram shows distribution of H3K4me2 lncRNAs with antisense (X) and sense (S) pattern with respect to their protein coding partner (pPCGs). ‘n’ denotes number of lncRNA-pPCG pairs in each pattern. ( E ) Genomic organization of H3K4me2 lncRNAs (green bars) with respect to nearest protein coding genes (grey bars). XH: where lncRNA shows Head-to-Head arrangement with nearby protein coding gene. XT: lncRNA in Tail-to-Tail arrangement with protein coding gene, XI: lncRNA located inside a protein coding gene, XO: lncRNA located outside and covers the entire protein coding gene. Sense pairs means lncRNAs in the same orientation as protein coding gene. The notion of greater or less than 2 kb means that the partner genes (pPCGs) are located within 2 kb (<2 kb) or away from 2 kb (> 2 kb) but within 50 kb window with respect to H3K4me2 lncRNAs. ( F ) Distribution of different patterns of genomic arrangements as described in (E) for H3K4me2 lncRNAs and non-CAR lncRNAs.

    Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

    Techniques: Transformation Assay, Expressing

    H3K4me2 XH lncRNAs regulate the transcriptional activity of their protein coding partners. ( A ) Nuclear expression levels of H3K4me2 XH lncRNAs and non-CAR XH lncRNAs. ( B ) Expression of H3K4me2 XH lncRNA-protein coding pairs (pPCGs) in nuclear input and H3K4me2 ChRIP samples. The P -value denotes the significance of difference between nuclear input and H3K4me2. ( C ) Gene ontology based functional enrichment analysis of protein coding genes that are partners (pPCGs) to H3K4me2 lncRNAs: divergent (XH), other antisense (XT, XI, and XO) and sense lncRNAs. The bar graph shows number of genes in corresponding ontology term and the heatmap represents the significance of individual terms ( P -values obtained using GeneSCF). The highlighted box, by the heatmap, is listed with different transcription factors from the enriched transcription related terms. ( D ) Gene expression analysis of three transcription factors ( FOXD3, GATA6 and HOXC13 ) using RT–qPCR analysis following downregulation of their neighboring H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) with siRNA in BT-549 cells. LncRNA expression is in green bars while transcription factors expression is in grey. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( E ) RT–qPCR analysis of FOXD3, GATA6 and HOXC13 gene expression after strand-specific downregulation of FOXD3-AS1, GATA6-AS1 and HOXC13-AS respectively, using LNA. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( F ) Gene expression analysis of three H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) using RT–qPCR analysis following downregulation of their respective transcription factor partners with siRNA or esiRNA in BT-549 cells. LncRNAs expression is depicted as green bars while transcription factors expression is in grey bars. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ns denotes non-significant.

    Journal: Nucleic Acids Research

    Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

    doi: 10.1093/nar/gky635

    Figure Lengend Snippet: H3K4me2 XH lncRNAs regulate the transcriptional activity of their protein coding partners. ( A ) Nuclear expression levels of H3K4me2 XH lncRNAs and non-CAR XH lncRNAs. ( B ) Expression of H3K4me2 XH lncRNA-protein coding pairs (pPCGs) in nuclear input and H3K4me2 ChRIP samples. The P -value denotes the significance of difference between nuclear input and H3K4me2. ( C ) Gene ontology based functional enrichment analysis of protein coding genes that are partners (pPCGs) to H3K4me2 lncRNAs: divergent (XH), other antisense (XT, XI, and XO) and sense lncRNAs. The bar graph shows number of genes in corresponding ontology term and the heatmap represents the significance of individual terms ( P -values obtained using GeneSCF). The highlighted box, by the heatmap, is listed with different transcription factors from the enriched transcription related terms. ( D ) Gene expression analysis of three transcription factors ( FOXD3, GATA6 and HOXC13 ) using RT–qPCR analysis following downregulation of their neighboring H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) with siRNA in BT-549 cells. LncRNA expression is in green bars while transcription factors expression is in grey. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( E ) RT–qPCR analysis of FOXD3, GATA6 and HOXC13 gene expression after strand-specific downregulation of FOXD3-AS1, GATA6-AS1 and HOXC13-AS respectively, using LNA. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( F ) Gene expression analysis of three H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) using RT–qPCR analysis following downregulation of their respective transcription factor partners with siRNA or esiRNA in BT-549 cells. LncRNAs expression is depicted as green bars while transcription factors expression is in grey bars. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ns denotes non-significant.

    Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

    Techniques: Activity Assay, Expressing, Functional Assay, Quantitative RT-PCR, esiRNA

    XH lncRNAs are overrepresented in active lncCARs. ( A ) Scatter plot showing enrichment of WDR5 lncRNAs over nuclear input. The X-axis denotes log transformed expression and Y-axis denotes log-fold change of WDR5 lncRNAs over nuclear input. Venn diagram, in the Scatter plot, shows 209 lncRNAs that are commonly enriched in both H3K4me2 and WDR5 ChRIP pulldowns. ( B ) Distribution of different patterns of genomic arrangements as described in Figure for WDR5 lncRNAs and non-CAR lncRNAs. ( C ) Distribution of different patterns of genomic arrangements as described in Figure for active lncRNA (ChRIP pulldown using H3K4me2 and WDR5 antibodies) and inactive lncRNA (ChRIP pulldown using EZH2 and H3K27me3 antibodies). ( D ) Nuclear expression levels of WDR5 XH lncRNAs, active XH lncCAR (WDR5 and H3K4me2) and non-CAR XH lncRNAs. ( E ) Enrichment of H3K4me2, H3K4me3, WDR5 ChIP-seq signals over active XH lncCAR (in green, n = 98) and non-CAR XH lncRNA (in black, n = 335) promoters (±2 kb) in BT-549 cells. The signals presented in the plots represent log 2 ratio between ChIP and input sample. TSS denotes the transcription start site of lncRNAs. ( F ) RT-qPCR analysis of WDR5 UV-RIP. The Y-axis shows fold enrichment relative to IgG. The significance of FOXD3-AS1 and HOXC13-AS enrichment in WDR5 RIP is calculated compared to the enrichment of FOXD3 and HOXC13 mRNAs, respectively. *** P ≤ 0.001. ( G ) Left panel: f-RIP of WDR5 WT and WDR5 F266A using FLAG antibody. The Y-axis shows percentage of Input. The significance of decrease in FOXD3-AS1, HOXC13-AS and positive control HOTTIP enrichment in WDR5 F266A RIP is calculated compared to their respective enrichments in WDR5-WT. GAPDH was used as negative control. Right panel: Expression of FLAG tagged WDR5 WT and WDR5 F266A mutant in HeLa cells by western blot. Data are shown as mean ± SD. ** P ≤ 0.01.

    Journal: Nucleic Acids Research

    Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

    doi: 10.1093/nar/gky635

    Figure Lengend Snippet: XH lncRNAs are overrepresented in active lncCARs. ( A ) Scatter plot showing enrichment of WDR5 lncRNAs over nuclear input. The X-axis denotes log transformed expression and Y-axis denotes log-fold change of WDR5 lncRNAs over nuclear input. Venn diagram, in the Scatter plot, shows 209 lncRNAs that are commonly enriched in both H3K4me2 and WDR5 ChRIP pulldowns. ( B ) Distribution of different patterns of genomic arrangements as described in Figure for WDR5 lncRNAs and non-CAR lncRNAs. ( C ) Distribution of different patterns of genomic arrangements as described in Figure for active lncRNA (ChRIP pulldown using H3K4me2 and WDR5 antibodies) and inactive lncRNA (ChRIP pulldown using EZH2 and H3K27me3 antibodies). ( D ) Nuclear expression levels of WDR5 XH lncRNAs, active XH lncCAR (WDR5 and H3K4me2) and non-CAR XH lncRNAs. ( E ) Enrichment of H3K4me2, H3K4me3, WDR5 ChIP-seq signals over active XH lncCAR (in green, n = 98) and non-CAR XH lncRNA (in black, n = 335) promoters (±2 kb) in BT-549 cells. The signals presented in the plots represent log 2 ratio between ChIP and input sample. TSS denotes the transcription start site of lncRNAs. ( F ) RT-qPCR analysis of WDR5 UV-RIP. The Y-axis shows fold enrichment relative to IgG. The significance of FOXD3-AS1 and HOXC13-AS enrichment in WDR5 RIP is calculated compared to the enrichment of FOXD3 and HOXC13 mRNAs, respectively. *** P ≤ 0.001. ( G ) Left panel: f-RIP of WDR5 WT and WDR5 F266A using FLAG antibody. The Y-axis shows percentage of Input. The significance of decrease in FOXD3-AS1, HOXC13-AS and positive control HOTTIP enrichment in WDR5 F266A RIP is calculated compared to their respective enrichments in WDR5-WT. GAPDH was used as negative control. Right panel: Expression of FLAG tagged WDR5 WT and WDR5 F266A mutant in HeLa cells by western blot. Data are shown as mean ± SD. ** P ≤ 0.01.

    Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

    Techniques: Transformation Assay, Expressing, ChIP-sequencing, Quantitative RT-PCR, Positive Control, Negative Control, Mutagenesis, Western Blot

    Active XH lncCARs promote transcription of their protein coding partners. ( A and B ) Promoter targeting of active XH lncCARs detected by ChOP. A) qPCR analysis of ChOP pull-downs, performed using FOXD3-AS1 and HOXC13-AS antisense probes, with primers over TSS (transcription start sites), regions spanning upstream and downstream of TSS and over transcription termination sites (TTS) of FOXD3 and HOXC13 genes in BT-549 cells. The ChOP pull-down with LacZ antisense oligo, used as a negative control (grey bars). Specific enrichment pattern for each of the XH lncCARs is depicted in green bars. The schematic in the right of the bar graph represents the genomic locations of the respective primers (grey: not enriched and rosetta: showing enrichment). Data are shown as mean ± SD (n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( B ) Similar qPCR analysis of ChOP pull-downs using FOXD3-AS1 and HOXC13-AS antisense probes upon ActD treatment in BT-549 cells. Data are shown as mean ± SD ( n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( C ) RT-qPCR analysis to detect relative levels of nascent FOXD3 and HOXC13 transcripts by Click-iT at 48 hours after the knockdown of FOXD3-AS1 and HOXC13-AS . Bar graph depicts the level of nascent protein coding transcripts upon removal of their respective active XH lncCARs. Data represent the mean ± SD of two independent biological experiments. * if P ≤ 0.05 and ** if P ≤ 0.01. ( D and E ) ChIP-qPCR analysis of the enrichment of H3K4me2 and H3K4me3 at the promoter region of FOXD3 (D) and HOXC13 (E) upon down regulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of H3K4me2 and H3K4me3 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA.The locations of ChIP primers used are depicted in schematic in the bottm of each bar graph. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( F and G ) ChIP-qPCR analysis of enrichment of WDR5 at the promoter region of FOXD3(F) and HOXC13 (G) upon downregulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of WDR5 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA. The ChIP primers used in the experiment are same as described in panel D–E. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( H and I ) FOXD3-AS1 and HOXC13-AS overexpression: RT–qPCR analysis of FOXD3 expression, upon ectopic overexpression of FOXD3-AS1 (H) and HOXC13-AS transcript (I) respectively, at Day 2 and Day 5 post transfection in BT-549 cell line. pcDNA empty vector transfection used as a control. Data represent the mean ± SD. of two independent biological experiments. P -values has been denoted as * if P ≤ 0.05, ** if P ≤ 0.01. ns denotes non-significant. ( J ) Cell fractionation in BT-549 cells show distribution of the overexpressed FOXD3-AS1 and HOXC13-AS transcripts. Data represent the mean ± SD of two independent biological experiments. GAPDH serves as positive control for cytoplasmic fraction, U6 and KCNQ1OT1 serves as positive control for nuclear fraction.

    Journal: Nucleic Acids Research

    Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

    doi: 10.1093/nar/gky635

    Figure Lengend Snippet: Active XH lncCARs promote transcription of their protein coding partners. ( A and B ) Promoter targeting of active XH lncCARs detected by ChOP. A) qPCR analysis of ChOP pull-downs, performed using FOXD3-AS1 and HOXC13-AS antisense probes, with primers over TSS (transcription start sites), regions spanning upstream and downstream of TSS and over transcription termination sites (TTS) of FOXD3 and HOXC13 genes in BT-549 cells. The ChOP pull-down with LacZ antisense oligo, used as a negative control (grey bars). Specific enrichment pattern for each of the XH lncCARs is depicted in green bars. The schematic in the right of the bar graph represents the genomic locations of the respective primers (grey: not enriched and rosetta: showing enrichment). Data are shown as mean ± SD (n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( B ) Similar qPCR analysis of ChOP pull-downs using FOXD3-AS1 and HOXC13-AS antisense probes upon ActD treatment in BT-549 cells. Data are shown as mean ± SD ( n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( C ) RT-qPCR analysis to detect relative levels of nascent FOXD3 and HOXC13 transcripts by Click-iT at 48 hours after the knockdown of FOXD3-AS1 and HOXC13-AS . Bar graph depicts the level of nascent protein coding transcripts upon removal of their respective active XH lncCARs. Data represent the mean ± SD of two independent biological experiments. * if P ≤ 0.05 and ** if P ≤ 0.01. ( D and E ) ChIP-qPCR analysis of the enrichment of H3K4me2 and H3K4me3 at the promoter region of FOXD3 (D) and HOXC13 (E) upon down regulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of H3K4me2 and H3K4me3 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA.The locations of ChIP primers used are depicted in schematic in the bottm of each bar graph. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( F and G ) ChIP-qPCR analysis of enrichment of WDR5 at the promoter region of FOXD3(F) and HOXC13 (G) upon downregulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of WDR5 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA. The ChIP primers used in the experiment are same as described in panel D–E. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( H and I ) FOXD3-AS1 and HOXC13-AS overexpression: RT–qPCR analysis of FOXD3 expression, upon ectopic overexpression of FOXD3-AS1 (H) and HOXC13-AS transcript (I) respectively, at Day 2 and Day 5 post transfection in BT-549 cell line. pcDNA empty vector transfection used as a control. Data represent the mean ± SD. of two independent biological experiments. P -values has been denoted as * if P ≤ 0.05, ** if P ≤ 0.01. ns denotes non-significant. ( J ) Cell fractionation in BT-549 cells show distribution of the overexpressed FOXD3-AS1 and HOXC13-AS transcripts. Data represent the mean ± SD of two independent biological experiments. GAPDH serves as positive control for cytoplasmic fraction, U6 and KCNQ1OT1 serves as positive control for nuclear fraction.

    Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

    Techniques: Negative Control, Quantitative RT-PCR, IF-P, Over Expression, Expressing, Transfection, Plasmid Preparation, Cell Fractionation, Positive Control