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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    Images

    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

    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: 92/100, 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
    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.
    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 "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

    bt 549 cells  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH bt 549 cells
    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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    bt549 cells  (CLS Cell Lines Service GmbH)


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


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    CLS Cell Lines Service GmbH bt 549 cells
    ( a ) RNA-fluorescence in situ hybridization showing the distribution of the MEG3 signal (green) in the nucleus (blue, stained with 4,6-diamidino-2-phenylindole). An RNase A-treated sample was used as a negative control. Scale bar, 1 μm. ( b ) RT–qPCR data showing the distribution of lncRNAs and protein-coding RNAs in the nuclear and cytoplasmic fractions (±s.d., n =3). ( c ) RT–qPCR analysis of MEG3 , KCNQ1OT1 and U1SnRNA in EZH2 RIP-purified RNA from <t>BT-549</t> cells. U1SnRNA served as negative control. The enrichment is plotted as percentage of input (±s.d., n =3). ( d ) Physical map of the MEG3 containing numbered exons showing two T-to-C transitions. The exons in red are constitutively expressed and blue are alternatively spliced exons. First conversion is part of exon 3 showing higher expression, whereas the second conversion is part of exon 4 showing low expression in the nuclear RNA sequencing. ( e ) In vitro interaction of MEG3 and PRC2. The schematic indicates the exons of the WT MEG3 clone. Left: RT–qPCR showing enrichment of sense WT MEG3 and MEG3 carrying deletions (Δ340-348 or Δ345-348 MEG3 ) in in vitro RNA binding assays. Reaction with antisense WT MEG3 or without purified PRC2 served as negative controls. The binding efficiency of MEG3 deletions were presented relative to WT MEG3 (±s.d., n =3). Right: RT–qPCR showing the quantification of input RNAs. ( f ) Upper panel: western blot showing EZH2 levels after pull-down with biotinylated sense WT MEG3 , antisense WT MEG3 , and Δ345-348 MEG3 RNAs incubated with nuclear extract. This is a representative data set from several experiments. Lower panel: agarose gel picture showing input biotin-RNA. ( g ) RT–qPCR result showing the relative enrichment of WT MEG3, Δ340-348 and Δ345-348 MEG3 RNAs in the EZH2-RIP, performed after BT-549 cells were transfected with WT and mutant MEG3 plasmids. Data were normalized to the input RNAs and plotted as percentage of input (±s.d., n =3). To distinguish the endogenous MEG3 from the ectopically expressed MEG3 , we designed RT–qPCRs primers, with one primer mapped to the transcribed portion of the vector and the other to MEG3 RNA. Endogenous MEG3 served as positive control and U1SnRNA as negative control.
    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 "MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA–DNA triplex structures"

    Article Title: MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA–DNA triplex structures

    Journal: Nature Communications

    doi: 10.1038/ncomms8743

    ( a ) RNA-fluorescence in situ hybridization showing the distribution of the MEG3 signal (green) in the nucleus (blue, stained with 4,6-diamidino-2-phenylindole). An RNase A-treated sample was used as a negative control. Scale bar, 1 μm. ( b ) RT–qPCR data showing the distribution of lncRNAs and protein-coding RNAs in the nuclear and cytoplasmic fractions (±s.d., n =3). ( c ) RT–qPCR analysis of MEG3 , KCNQ1OT1 and U1SnRNA in EZH2 RIP-purified RNA from BT-549 cells. U1SnRNA served as negative control. The enrichment is plotted as percentage of input (±s.d., n =3). ( d ) Physical map of the MEG3 containing numbered exons showing two T-to-C transitions. The exons in red are constitutively expressed and blue are alternatively spliced exons. First conversion is part of exon 3 showing higher expression, whereas the second conversion is part of exon 4 showing low expression in the nuclear RNA sequencing. ( e ) In vitro interaction of MEG3 and PRC2. The schematic indicates the exons of the WT MEG3 clone. Left: RT–qPCR showing enrichment of sense WT MEG3 and MEG3 carrying deletions (Δ340-348 or Δ345-348 MEG3 ) in in vitro RNA binding assays. Reaction with antisense WT MEG3 or without purified PRC2 served as negative controls. The binding efficiency of MEG3 deletions were presented relative to WT MEG3 (±s.d., n =3). Right: RT–qPCR showing the quantification of input RNAs. ( f ) Upper panel: western blot showing EZH2 levels after pull-down with biotinylated sense WT MEG3 , antisense WT MEG3 , and Δ345-348 MEG3 RNAs incubated with nuclear extract. This is a representative data set from several experiments. Lower panel: agarose gel picture showing input biotin-RNA. ( g ) RT–qPCR result showing the relative enrichment of WT MEG3, Δ340-348 and Δ345-348 MEG3 RNAs in the EZH2-RIP, performed after BT-549 cells were transfected with WT and mutant MEG3 plasmids. Data were normalized to the input RNAs and plotted as percentage of input (±s.d., n =3). To distinguish the endogenous MEG3 from the ectopically expressed MEG3 , we designed RT–qPCRs primers, with one primer mapped to the transcribed portion of the vector and the other to MEG3 RNA. Endogenous MEG3 served as positive control and U1SnRNA as negative control.
    Figure Legend Snippet: ( a ) RNA-fluorescence in situ hybridization showing the distribution of the MEG3 signal (green) in the nucleus (blue, stained with 4,6-diamidino-2-phenylindole). An RNase A-treated sample was used as a negative control. Scale bar, 1 μm. ( b ) RT–qPCR data showing the distribution of lncRNAs and protein-coding RNAs in the nuclear and cytoplasmic fractions (±s.d., n =3). ( c ) RT–qPCR analysis of MEG3 , KCNQ1OT1 and U1SnRNA in EZH2 RIP-purified RNA from BT-549 cells. U1SnRNA served as negative control. The enrichment is plotted as percentage of input (±s.d., n =3). ( d ) Physical map of the MEG3 containing numbered exons showing two T-to-C transitions. The exons in red are constitutively expressed and blue are alternatively spliced exons. First conversion is part of exon 3 showing higher expression, whereas the second conversion is part of exon 4 showing low expression in the nuclear RNA sequencing. ( e ) In vitro interaction of MEG3 and PRC2. The schematic indicates the exons of the WT MEG3 clone. Left: RT–qPCR showing enrichment of sense WT MEG3 and MEG3 carrying deletions (Δ340-348 or Δ345-348 MEG3 ) in in vitro RNA binding assays. Reaction with antisense WT MEG3 or without purified PRC2 served as negative controls. The binding efficiency of MEG3 deletions were presented relative to WT MEG3 (±s.d., n =3). Right: RT–qPCR showing the quantification of input RNAs. ( f ) Upper panel: western blot showing EZH2 levels after pull-down with biotinylated sense WT MEG3 , antisense WT MEG3 , and Δ345-348 MEG3 RNAs incubated with nuclear extract. This is a representative data set from several experiments. Lower panel: agarose gel picture showing input biotin-RNA. ( g ) RT–qPCR result showing the relative enrichment of WT MEG3, Δ340-348 and Δ345-348 MEG3 RNAs in the EZH2-RIP, performed after BT-549 cells were transfected with WT and mutant MEG3 plasmids. Data were normalized to the input RNAs and plotted as percentage of input (±s.d., n =3). To distinguish the endogenous MEG3 from the ectopically expressed MEG3 , we designed RT–qPCRs primers, with one primer mapped to the transcribed portion of the vector and the other to MEG3 RNA. Endogenous MEG3 served as positive control and U1SnRNA as negative control.

    Techniques Used: Fluorescence, In Situ Hybridization, Staining, Negative Control, Quantitative RT-PCR, Purification, Expressing, RNA Sequencing Assay, In Vitro, RNA Binding Assay, Binding Assay, Western Blot, Incubation, Agarose Gel Electrophoresis, Transfection, Mutagenesis, Plasmid Preparation, Positive Control

    ( a–c ) MEG3 and EZH2 share common gene targets. ( a ) Venn diagram showing the number of genes deregulated after downregulation of MEG3 and EZH2 using siRNA in BT-549 and HF cells, and the degree of overlap between the MEG3- and EZH2-dependent genes. The P values were obtained by hypergeometric test using all protein-coding genes as a background. ( b ) EZH2 protein levels, as determined by western blotting, following EZH2 and MEG3 downregulation in BT-549 and HF cells. Tubulin was used as a loading control. ( c ) RT–qPCR analysis of EZH2 and MEG3 mRNA expression in Ctrlsi, EZH2 si and MEG3 si transfected BT-549 and HF cells (±s.d., n =3). ( d ) RT–qPCR analysis of TGFB2 , TGFBR1 and SMAD2 gene expression in Ctrlsi, MEG3 si and EZH2 si transfected BT-549 cells (±s.d., n =3). ( e ) Immunoblot showing SMAD2, TGFBR1 and tubulin protein levels following transfection of BT-549 cells with Ctrlsi and MEG3 si. ( f ) Bar graph showing RT–qPCR analysis of TGFB2 , TGFBR1 and SMAD2 mRNA levels after overexpression of MEG3 (pREP4 MEG3 ) in BT-549 and MDA-MB-231 cells. The levels in pREP4 MEG3 are presented relative to CtrlpREP4 (±s.d., n =3). EZH2 was used as a control showing no change in expression after overexpression of MEG3 . The P values were calculated using Student's t -test (two-tailed, two-sample unequal variance), * P <0.05. ( g ) Downregulation of MEG3 influences the invasive property of BT-549 cells through regulation of the TGF-β pathway. Images showing Matrigel invasion of the BT-549 cells. The two images in the upper panel show invasion of BT-549 cells infected with Ctrlsh lentivirus, and Ctrlsh infection followed by incubation with TGF-β2 ligand (Ctrlsh-TGFB2). The images in the bottom panel show the cells infected with MEG3 Sh and MEG3 sh infection followed by incubation with TGF-β inhibitor ( MEG3 sh+ TGF-β in). Scale bar, 10 μm. The bar graph shows quantification (±s.d., n =3) of the matrix invaded cells in MEG3 sh relative to the Ctrlsh. The P values were calculated using Student's t -test (two-tailed, two-sample unequal variance), * P <0.05, ** P <0.01.
    Figure Legend Snippet: ( a–c ) MEG3 and EZH2 share common gene targets. ( a ) Venn diagram showing the number of genes deregulated after downregulation of MEG3 and EZH2 using siRNA in BT-549 and HF cells, and the degree of overlap between the MEG3- and EZH2-dependent genes. The P values were obtained by hypergeometric test using all protein-coding genes as a background. ( b ) EZH2 protein levels, as determined by western blotting, following EZH2 and MEG3 downregulation in BT-549 and HF cells. Tubulin was used as a loading control. ( c ) RT–qPCR analysis of EZH2 and MEG3 mRNA expression in Ctrlsi, EZH2 si and MEG3 si transfected BT-549 and HF cells (±s.d., n =3). ( d ) RT–qPCR analysis of TGFB2 , TGFBR1 and SMAD2 gene expression in Ctrlsi, MEG3 si and EZH2 si transfected BT-549 cells (±s.d., n =3). ( e ) Immunoblot showing SMAD2, TGFBR1 and tubulin protein levels following transfection of BT-549 cells with Ctrlsi and MEG3 si. ( f ) Bar graph showing RT–qPCR analysis of TGFB2 , TGFBR1 and SMAD2 mRNA levels after overexpression of MEG3 (pREP4 MEG3 ) in BT-549 and MDA-MB-231 cells. The levels in pREP4 MEG3 are presented relative to CtrlpREP4 (±s.d., n =3). EZH2 was used as a control showing no change in expression after overexpression of MEG3 . The P values were calculated using Student's t -test (two-tailed, two-sample unequal variance), * P <0.05. ( g ) Downregulation of MEG3 influences the invasive property of BT-549 cells through regulation of the TGF-β pathway. Images showing Matrigel invasion of the BT-549 cells. The two images in the upper panel show invasion of BT-549 cells infected with Ctrlsh lentivirus, and Ctrlsh infection followed by incubation with TGF-β2 ligand (Ctrlsh-TGFB2). The images in the bottom panel show the cells infected with MEG3 Sh and MEG3 sh infection followed by incubation with TGF-β inhibitor ( MEG3 sh+ TGF-β in). Scale bar, 10 μm. The bar graph shows quantification (±s.d., n =3) of the matrix invaded cells in MEG3 sh relative to the Ctrlsh. The P values were calculated using Student's t -test (two-tailed, two-sample unequal variance), * P <0.05, ** P <0.01.

    Techniques Used: Western Blot, Quantitative RT-PCR, Expressing, Transfection, Over Expression, Two Tailed Test, Infection, Incubation

    KEGG pathway analysis of the deregulated genes identified by microarray and RNA-sequencing after downregulation of MEG3 and EZH2 by siRNA in  BT-549 cells  using GeneSCF.
    Figure Legend Snippet: KEGG pathway analysis of the deregulated genes identified by microarray and RNA-sequencing after downregulation of MEG3 and EZH2 by siRNA in BT-549 cells using GeneSCF.

    Techniques Used: Microarray, Infection

    Summary of MEG3 peaks with associated genes and their overlap with H3K4me1 peaks in  BT-549 cells.
    Figure Legend Snippet: Summary of MEG3 peaks with associated genes and their overlap with H3K4me1 peaks in BT-549 cells.

    Techniques Used: Binding Assay

    ( a ) RT–qPCR analysis showing specific enrichment (presented as percentage of input) of MEG3 but not MALAT1 RNA in the ChOP pull-down assay with MEG3 antisense probes. The ChOP pull-down with GFP antisense oligo, used as a negative control, did not show any enrichment of MEG3 and MALAT1 RNAs. ( b ) Genomic tracks showing ChOP-seq ( MEG3 , GFP and input) and ChIP-seq (H3K4me1) intensities, visualized in log scale. The MEG3 binding site is located upstream of the TGFBR1 gene (falls within the intron of the COL15A1 gene) and it overlaps with H3K4me1 peaks in BT-549 cells. ( c ) ChIP–qPCR showing enrichment of H3K27me3 chromatin marks, presented as percentage of input, over the MEG3 peaks associated with the TGF-β genes in Ctrlsh and MEG3 sh cells (±s.d., n =3). ( d ) Schematic outline of the TGFBR1 gene showing MEG3 peaks and the location of 3C primers (P1–P9), as indicated by arrows. EcoRI restriction sites are shown as blue vertical lines. Each error bar represents ±s.d. from three experiments. Looping events between the upstream MEG3 binding site (corresponding to P2 primer) and the TGFBR1 promoter detected by 3C–qPCR in Ctrlsh and MEG3 sh cells. The P values were calculated using Student's t -test (two-tailed, two-sample unequal variance), * P <0.05.
    Figure Legend Snippet: ( a ) RT–qPCR analysis showing specific enrichment (presented as percentage of input) of MEG3 but not MALAT1 RNA in the ChOP pull-down assay with MEG3 antisense probes. The ChOP pull-down with GFP antisense oligo, used as a negative control, did not show any enrichment of MEG3 and MALAT1 RNAs. ( b ) Genomic tracks showing ChOP-seq ( MEG3 , GFP and input) and ChIP-seq (H3K4me1) intensities, visualized in log scale. The MEG3 binding site is located upstream of the TGFBR1 gene (falls within the intron of the COL15A1 gene) and it overlaps with H3K4me1 peaks in BT-549 cells. ( c ) ChIP–qPCR showing enrichment of H3K27me3 chromatin marks, presented as percentage of input, over the MEG3 peaks associated with the TGF-β genes in Ctrlsh and MEG3 sh cells (±s.d., n =3). ( d ) Schematic outline of the TGFBR1 gene showing MEG3 peaks and the location of 3C primers (P1–P9), as indicated by arrows. EcoRI restriction sites are shown as blue vertical lines. Each error bar represents ±s.d. from three experiments. Looping events between the upstream MEG3 binding site (corresponding to P2 primer) and the TGFBR1 promoter detected by 3C–qPCR in Ctrlsh and MEG3 sh cells. The P values were calculated using Student's t -test (two-tailed, two-sample unequal variance), * P <0.05.

    Techniques Used: Quantitative RT-PCR, Pull Down Assay, Negative Control, ChIP-sequencing, Binding Assay, Two Tailed Test

    ( a ) Predicted GA-rich motifs enriched in all MEG3 peaks and peaks associated with deregulated genes using MEME-ChIP. ( b ) Number of TrTS over the MEG3 peak summits and neighbouring regions, predicted by Triplexator . ( c ) The schematic shows the MEG3 TFO used in the triplex assays. The exons are colour-coded as described before. ( d ) Electrophoretic mobility shift assay. End-labelled dsDNA oligos (sequences provided in the schematic with gene name) were incubated alone (lane 1) or with increasing concentrations of MEG3 ssRNA TFO (lanes 2 and 3: shift indicated with arrow) or with increasing concentrations of control ssRNA oligo (lanes 8 and 9). dsDNA oligos were incubated with MEG3 TFO and treated with either RNase A (lane 4) or RNase H (lane 5). dsDNA oligos were incubated with MEG3 ssRNA TFO in the presence of either unlabelled specific competitor (lane 6) or nonspecific competitor (lane 7). ( e ) TGFBR1- associated MEG3 peak sequence and its mutated version (the changed nucleotides are in red) were incubated alone (lanes 1 and 7) or with MEG3 TFO. Arrow indicates complex formation. ( f , g ) Enrichment of MEG3 peak sequences using biotin- and psoralen-labelled MEG3 TFO. RNase H-treated lysates were used to capture the labelled MEG3 TFO using streptavidin beads. The enrichment of MEG3 peaks is presented as the ratio between MEG3 TFO and control oligo (±s.d., n =3). ( h ) RT–qPCR analysis of gene expression in BT-549 cells transfected with either MEG3 TFO or control RNA oligo. Expression in MEG3 TFO presented relative to the control oligo (±s.d., n =3). * P <0.05, Student's t -test (two-tailed, two-sample unequal variance). ( i ) Left panel: CD spectra of a 1:1 mixture of TGFB2 dsDNA and MEG3 TFO (ssRNA) are shown in black, and TGFB2 dsDNA and the control ssRNA are shown in red. Right panel: the sum of the individual CD spectra for TGFB2 dsDNA and MEG3 TFO (ssRNA) is shown in black, and the sum of the individual CD spectra for TGFB2 dsDNA and the control ssRNA is shown in red. Inset in the left and right panel shows the difference between the two spectra.
    Figure Legend Snippet: ( a ) Predicted GA-rich motifs enriched in all MEG3 peaks and peaks associated with deregulated genes using MEME-ChIP. ( b ) Number of TrTS over the MEG3 peak summits and neighbouring regions, predicted by Triplexator . ( c ) The schematic shows the MEG3 TFO used in the triplex assays. The exons are colour-coded as described before. ( d ) Electrophoretic mobility shift assay. End-labelled dsDNA oligos (sequences provided in the schematic with gene name) were incubated alone (lane 1) or with increasing concentrations of MEG3 ssRNA TFO (lanes 2 and 3: shift indicated with arrow) or with increasing concentrations of control ssRNA oligo (lanes 8 and 9). dsDNA oligos were incubated with MEG3 TFO and treated with either RNase A (lane 4) or RNase H (lane 5). dsDNA oligos were incubated with MEG3 ssRNA TFO in the presence of either unlabelled specific competitor (lane 6) or nonspecific competitor (lane 7). ( e ) TGFBR1- associated MEG3 peak sequence and its mutated version (the changed nucleotides are in red) were incubated alone (lanes 1 and 7) or with MEG3 TFO. Arrow indicates complex formation. ( f , g ) Enrichment of MEG3 peak sequences using biotin- and psoralen-labelled MEG3 TFO. RNase H-treated lysates were used to capture the labelled MEG3 TFO using streptavidin beads. The enrichment of MEG3 peaks is presented as the ratio between MEG3 TFO and control oligo (±s.d., n =3). ( h ) RT–qPCR analysis of gene expression in BT-549 cells transfected with either MEG3 TFO or control RNA oligo. Expression in MEG3 TFO presented relative to the control oligo (±s.d., n =3). * P <0.05, Student's t -test (two-tailed, two-sample unequal variance). ( i ) Left panel: CD spectra of a 1:1 mixture of TGFB2 dsDNA and MEG3 TFO (ssRNA) are shown in black, and TGFB2 dsDNA and the control ssRNA are shown in red. Right panel: the sum of the individual CD spectra for TGFB2 dsDNA and MEG3 TFO (ssRNA) is shown in black, and the sum of the individual CD spectra for TGFB2 dsDNA and the control ssRNA is shown in red. Inset in the left and right panel shows the difference between the two spectra.

    Techniques Used: Electrophoretic Mobility Shift Assay, Incubation, Sequencing, Quantitative RT-PCR, Expressing, Transfection, Two Tailed Test

    ( a ) Confocal microscopic images showing immunostaining with anti-triplex dA.2rU antibody (green) in BT-549 cells. The nucleus is stained with DAPI (4,6-diamidino-2-phenylindole; blue). Immunostaining with no antibody and secondary antibody were used as negative controls. Scale bar, 5 μm. The graph to the right shows quantification of the triplex signal in cytoplasm and nuclear compartments obtained from the three-dimensional confocal images. The graph represents the average of cytoplasmic and nuclear signals from >50 cells in several microscopic fields. The error bars indicate s.e.m. The P value was calculated using Student's t -test ** P <0.01. ( b ) RNA–DNA triplex structures are sensitive to RNase A but are resistant to RNase H in vivo . Top panel: immunofluorescent staining of BT-549 cells with anti-triplex dA.2rU antibody (green) with no treatment (left), pretreated with RNase A (centre), or pretreated with RNase H (right) as indicated. Middle panel: cells were counterstained with DAPI (blue). Bottom panel: overlay of the triplex signals with DAPI staining. Scale bar, 5 μm. ( c ) Triplex-ChIP–qPCR showing enrichment (presented as percentage of input) of triplex structures over the MEG3 peaks associated with the TGF-β pathway genes (TGFBR1 , TGFB2 and SMAD2 ) in BT-549 cells (±s.d., n =3). Actin was used as a negative control. Chromatin was pretreated with RNase A or RNase H before ChIP. Immunoglobulin G (IgG) was used as an antibody control. ( d ) Triplex-ChIP–qPCR showing enrichment (presented as percentage of input) of triplex structures over the MEG3 peaks associated with the TGF-β pathway genes (TGFBR1 , TGFB2 and SMAD2 ) in Ctrlsh and MEG3 sh BT-549 cells (±s.d., n =3). IgG was used as an antibody control.
    Figure Legend Snippet: ( a ) Confocal microscopic images showing immunostaining with anti-triplex dA.2rU antibody (green) in BT-549 cells. The nucleus is stained with DAPI (4,6-diamidino-2-phenylindole; blue). Immunostaining with no antibody and secondary antibody were used as negative controls. Scale bar, 5 μm. The graph to the right shows quantification of the triplex signal in cytoplasm and nuclear compartments obtained from the three-dimensional confocal images. The graph represents the average of cytoplasmic and nuclear signals from >50 cells in several microscopic fields. The error bars indicate s.e.m. The P value was calculated using Student's t -test ** P <0.01. ( b ) RNA–DNA triplex structures are sensitive to RNase A but are resistant to RNase H in vivo . Top panel: immunofluorescent staining of BT-549 cells with anti-triplex dA.2rU antibody (green) with no treatment (left), pretreated with RNase A (centre), or pretreated with RNase H (right) as indicated. Middle panel: cells were counterstained with DAPI (blue). Bottom panel: overlay of the triplex signals with DAPI staining. Scale bar, 5 μm. ( c ) Triplex-ChIP–qPCR showing enrichment (presented as percentage of input) of triplex structures over the MEG3 peaks associated with the TGF-β pathway genes (TGFBR1 , TGFB2 and SMAD2 ) in BT-549 cells (±s.d., n =3). Actin was used as a negative control. Chromatin was pretreated with RNase A or RNase H before ChIP. Immunoglobulin G (IgG) was used as an antibody control. ( d ) Triplex-ChIP–qPCR showing enrichment (presented as percentage of input) of triplex structures over the MEG3 peaks associated with the TGF-β pathway genes (TGFBR1 , TGFB2 and SMAD2 ) in Ctrlsh and MEG3 sh BT-549 cells (±s.d., n =3). IgG was used as an antibody control.

    Techniques Used: Immunostaining, Staining, In Vivo, Negative Control

    ( a ) MEG3 -PRC2 in vitro binding assay. Left panel: schematic representation of WT MEG3 , Δ46-56 MEG3 and Δ345-348 MEG3 . Green and red boxes indicate PRC2- and chromatin-interacting sequences, respectively. Middle panel: bar diagram showing the relative binding efficiency (as determined by RT–qPCR) of the sense WT MEG3 , Δ46-56 MEG3 and Δ345-348 MEG3 RNAs in an in vitro PRC2-binding assay. Binding assays with no PRC2 and antisense WT MEG3 served as negative controls. The PRC2-binding efficiency of sense WT MEG3 was set to 100, and the binding efficiency of the MEG3 mutants is presented relative to WT MEG3 (±s.d., n =3). Right panel: RT–qPCR showing the quantification of the input sense WT MEG3 , MEG3 deletions (Δ340-348 or Δ345-348 MEG3) and antisense WT MEG3 RNAs. ( b ) Deletion of MEG3 TFO leads to loss of chromatin interaction. Left panel: schematic display of interaction of the WT MEG3 and MEG3 mutants (Δ46-56 MEG3 and Δ345-348 MEG3 ) with the MEG3 peak sequences in vivo . Red ( MEG3 TFO) and green (PRC2-interacting region) colour-coded regions indicate the location of the deleted MEG3 RNA sequences 46-56 and 345-348, respectively. Biotin-labelled WT MEG3 or MEG3 mutants were used to transfect BT-549 cells followed by crosslinking with formaldehyde. RNAse H-treated cell lysates were incubated with streptavidin beads to capture the MEG3 RNA-associated DNA. Middle panel: qPCR data are presented as the ratio of captured DNA in WT MEG3 or MEG3 mutants to captured non-biotinylated MEG3 RNA (±s.d., n =3). Right panel: agarose gel picture showing the quality of the biotin-labelled WT and mutant MEG3 RNAs (500 ng of each biotin-RNA was loaded). ( c ) Model depicting how chromatin-interacting sequences of MEG3 lncRNA-containing GA-rich sequences form RNA–DNA triplex with the GA-rich DNA sequences to guide MEG3 lncRNA to chromatin. PRC2-interacting sequences of MEG3 lncRNA facilitate recruitment of the PRC2 to distal regulatory elements, thereby establishing H3K27me3 marks to modulate gene expression.
    Figure Legend Snippet: ( a ) MEG3 -PRC2 in vitro binding assay. Left panel: schematic representation of WT MEG3 , Δ46-56 MEG3 and Δ345-348 MEG3 . Green and red boxes indicate PRC2- and chromatin-interacting sequences, respectively. Middle panel: bar diagram showing the relative binding efficiency (as determined by RT–qPCR) of the sense WT MEG3 , Δ46-56 MEG3 and Δ345-348 MEG3 RNAs in an in vitro PRC2-binding assay. Binding assays with no PRC2 and antisense WT MEG3 served as negative controls. The PRC2-binding efficiency of sense WT MEG3 was set to 100, and the binding efficiency of the MEG3 mutants is presented relative to WT MEG3 (±s.d., n =3). Right panel: RT–qPCR showing the quantification of the input sense WT MEG3 , MEG3 deletions (Δ340-348 or Δ345-348 MEG3) and antisense WT MEG3 RNAs. ( b ) Deletion of MEG3 TFO leads to loss of chromatin interaction. Left panel: schematic display of interaction of the WT MEG3 and MEG3 mutants (Δ46-56 MEG3 and Δ345-348 MEG3 ) with the MEG3 peak sequences in vivo . Red ( MEG3 TFO) and green (PRC2-interacting region) colour-coded regions indicate the location of the deleted MEG3 RNA sequences 46-56 and 345-348, respectively. Biotin-labelled WT MEG3 or MEG3 mutants were used to transfect BT-549 cells followed by crosslinking with formaldehyde. RNAse H-treated cell lysates were incubated with streptavidin beads to capture the MEG3 RNA-associated DNA. Middle panel: qPCR data are presented as the ratio of captured DNA in WT MEG3 or MEG3 mutants to captured non-biotinylated MEG3 RNA (±s.d., n =3). Right panel: agarose gel picture showing the quality of the biotin-labelled WT and mutant MEG3 RNAs (500 ng of each biotin-RNA was loaded). ( c ) Model depicting how chromatin-interacting sequences of MEG3 lncRNA-containing GA-rich sequences form RNA–DNA triplex with the GA-rich DNA sequences to guide MEG3 lncRNA to chromatin. PRC2-interacting sequences of MEG3 lncRNA facilitate recruitment of the PRC2 to distal regulatory elements, thereby establishing H3K27me3 marks to modulate gene expression.

    Techniques Used: In Vitro, Binding Assay, Quantitative RT-PCR, In Vivo, Incubation, Agarose Gel Electrophoresis, Mutagenesis, Expressing

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    CLS Cell Lines Service GmbH bt 549 cells
    A: The effect of MTHFD2 siRNAs on MDA-MB-231(SA) and <t>BT-549</t> cell motility in wound healing experiment. Cells were automatically imaged once every hour after wound scratching. Wound closure effect was calculated as wound confluence which the cells gained in twelve hours. B: MDA-MB-231(SA) cells transfected with either vimentin targeting siRNA (siVIM), MTHFD2 targeting siRNA (siMTHFD2) or negative control siRNA (siScr), allowed to invade into Matrigel matrix for 4 days and imaged. Shown are the quantifications of two replicate experiments (imaged at 5-10 different positions; ***p < 0.005) and representative images. The dashed lines indicate the top of the Matrigel matrix.
    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 "High-throughput RNAi screening for novel modulators of vimentin expression identifies MTHFD2 as a regulator of breast cancer cell migration and invasion"

    Article Title: High-throughput RNAi screening for novel modulators of vimentin expression identifies MTHFD2 as a regulator of breast cancer cell migration and invasion

    Journal: Oncotarget

    doi:

    A: The effect of MTHFD2 siRNAs on MDA-MB-231(SA) and BT-549 cell motility in wound healing experiment. Cells were automatically imaged once every hour after wound scratching. Wound closure effect was calculated as wound confluence which the cells gained in twelve hours. B: MDA-MB-231(SA) cells transfected with either vimentin targeting siRNA (siVIM), MTHFD2 targeting siRNA (siMTHFD2) or negative control siRNA (siScr), allowed to invade into Matrigel matrix for 4 days and imaged. Shown are the quantifications of two replicate experiments (imaged at 5-10 different positions; ***p < 0.005) and representative images. The dashed lines indicate the top of the Matrigel matrix.
    Figure Legend Snippet: A: The effect of MTHFD2 siRNAs on MDA-MB-231(SA) and BT-549 cell motility in wound healing experiment. Cells were automatically imaged once every hour after wound scratching. Wound closure effect was calculated as wound confluence which the cells gained in twelve hours. B: MDA-MB-231(SA) cells transfected with either vimentin targeting siRNA (siVIM), MTHFD2 targeting siRNA (siMTHFD2) or negative control siRNA (siScr), allowed to invade into Matrigel matrix for 4 days and imaged. Shown are the quantifications of two replicate experiments (imaged at 5-10 different positions; ***p < 0.005) and representative images. The dashed lines indicate the top of the Matrigel matrix.

    Techniques Used: Transfection, Negative Control

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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.
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    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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    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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    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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    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