53bp1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 53bp1
    Recruitment of various DDR proteins to lacO arrays in U2OS 40–2-6 ER-LacI cells. (A) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT for 2 h were double immunostained with the indicated antibodies and counterstained with DAPI. ssDNA was stained with anti-BrdU antibody under nondenaturing conditions. For detection of total ssDNA, DNA was labeled for 24 h with 10 µM BrdU. For detection of nascent-strand ssDNA, newly synthesized DNA was labeled with BrdU during treatment with 4-OHT. For detection of parental-strand ssDNA, DNA was labeled with 10 µM BrdU for 20 h, followed by a chase in fresh medium without BrdU for 2 h before addition of 4-OHT. Representative images are shown, and the colocalization frequencies are shown in . Yellow arrows indicate colocalization of DDR proteins with the foci of HA-LacI, and white arrows indicate noncolocalization. Scale bars, 10 µm. (B) The antibody used in this study can detect bleomycin-induced RAD51 foci. U2OS 40–2-6 ER-LacI cells were treated with 8 µM Bleomycin for 18 h, then immunostained with anti-RAD51 antibody, followed by DAPI staining. Representative images are shown. Scale bar, 10 µm. (C) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT or a control vehicle (EtOH) for 2 h were subjected to ChIP-quantitative PCR analysis using the indicated antibodies. Enrichment of the lacO sequences is shown as the percent of input DNA. Means ± SD are shown ( n = 6 for control rabbit IgG; n = 7 for LacI and <t>53BP1).</t> *, P < 0.05; ***, P < 0.001; n.s., not significant (two-tailed Student’s t test).
    53bp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/53bp1/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    53bp1 - by Bioz Stars, 2023-01
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    Images

    1) Product Images from "SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions"

    Article Title: SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.202003148

    Recruitment of various DDR proteins to lacO arrays in U2OS 40–2-6 ER-LacI cells. (A) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT for 2 h were double immunostained with the indicated antibodies and counterstained with DAPI. ssDNA was stained with anti-BrdU antibody under nondenaturing conditions. For detection of total ssDNA, DNA was labeled for 24 h with 10 µM BrdU. For detection of nascent-strand ssDNA, newly synthesized DNA was labeled with BrdU during treatment with 4-OHT. For detection of parental-strand ssDNA, DNA was labeled with 10 µM BrdU for 20 h, followed by a chase in fresh medium without BrdU for 2 h before addition of 4-OHT. Representative images are shown, and the colocalization frequencies are shown in . Yellow arrows indicate colocalization of DDR proteins with the foci of HA-LacI, and white arrows indicate noncolocalization. Scale bars, 10 µm. (B) The antibody used in this study can detect bleomycin-induced RAD51 foci. U2OS 40–2-6 ER-LacI cells were treated with 8 µM Bleomycin for 18 h, then immunostained with anti-RAD51 antibody, followed by DAPI staining. Representative images are shown. Scale bar, 10 µm. (C) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT or a control vehicle (EtOH) for 2 h were subjected to ChIP-quantitative PCR analysis using the indicated antibodies. Enrichment of the lacO sequences is shown as the percent of input DNA. Means ± SD are shown ( n = 6 for control rabbit IgG; n = 7 for LacI and 53BP1). *, P < 0.05; ***, P < 0.001; n.s., not significant (two-tailed Student’s t test).
    Figure Legend Snippet: Recruitment of various DDR proteins to lacO arrays in U2OS 40–2-6 ER-LacI cells. (A) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT for 2 h were double immunostained with the indicated antibodies and counterstained with DAPI. ssDNA was stained with anti-BrdU antibody under nondenaturing conditions. For detection of total ssDNA, DNA was labeled for 24 h with 10 µM BrdU. For detection of nascent-strand ssDNA, newly synthesized DNA was labeled with BrdU during treatment with 4-OHT. For detection of parental-strand ssDNA, DNA was labeled with 10 µM BrdU for 20 h, followed by a chase in fresh medium without BrdU for 2 h before addition of 4-OHT. Representative images are shown, and the colocalization frequencies are shown in . Yellow arrows indicate colocalization of DDR proteins with the foci of HA-LacI, and white arrows indicate noncolocalization. Scale bars, 10 µm. (B) The antibody used in this study can detect bleomycin-induced RAD51 foci. U2OS 40–2-6 ER-LacI cells were treated with 8 µM Bleomycin for 18 h, then immunostained with anti-RAD51 antibody, followed by DAPI staining. Representative images are shown. Scale bar, 10 µm. (C) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT or a control vehicle (EtOH) for 2 h were subjected to ChIP-quantitative PCR analysis using the indicated antibodies. Enrichment of the lacO sequences is shown as the percent of input DNA. Means ± SD are shown ( n = 6 for control rabbit IgG; n = 7 for LacI and 53BP1). *, P < 0.05; ***, P < 0.001; n.s., not significant (two-tailed Student’s t test).

    Techniques Used: Staining, Labeling, Synthesized, Real-time Polymerase Chain Reaction, Two Tailed Test

    A model of the fate of underreplicated DNA regions in the lacO array. Even in the absence of LacI binding, the lacO array is a difficult-to-replicate region, like other repeat sequences, and contains underreplicated lesions (∼60% on the basis of the data shown in ), which persist until late G2/M phase. Replication stress by LacI binding to the array further increases the frequency of cells with underreplicated DNA lesions (∼90%). To try to complete replication of the array during S phase, cells activate the DDR, in which one-ended DSB is first generated by SLX4–XPF–mediated DNA cleavage. Excess ssDNA is then generated by unidentified exonucleases, which in turn recruit ATR, FANCD2, and RAD52. ATR and FANCD2 are interdependently recruited. The DDR may promote completion of replication, because SLX4 and ATR inhibition exacerbates mitotic abnormality induced by LacI. The underreplicated intermediates persisting until late G2/M phase are processed by MUS81-mediated cleavage in early mitosis. Note that cleaved strands are arbitrary in the figure, because it remains unclear whether the cleavage occurs in the leading or lagging strand templates. Cleavage at lacO can promote MiDAS (∼20% in the absence or presence of LacI; ) or end joining that results in deletion of the loci (∼30% in the absence of LacI and ∼50% in the presence of LacI; ). A fraction of the lacO arrays escape from the cleavage and remain underreplicated until anaphase, leading to the anaphase abnormal lacO structure (∼5% in the absence of LacI and ∼20% in the presence of LacI; ) and the formation of 53BP1 NBs in daughter G1 cells (∼5% in the absence of LacI and ∼20% in the presence of LacI; ).
    Figure Legend Snippet: A model of the fate of underreplicated DNA regions in the lacO array. Even in the absence of LacI binding, the lacO array is a difficult-to-replicate region, like other repeat sequences, and contains underreplicated lesions (∼60% on the basis of the data shown in ), which persist until late G2/M phase. Replication stress by LacI binding to the array further increases the frequency of cells with underreplicated DNA lesions (∼90%). To try to complete replication of the array during S phase, cells activate the DDR, in which one-ended DSB is first generated by SLX4–XPF–mediated DNA cleavage. Excess ssDNA is then generated by unidentified exonucleases, which in turn recruit ATR, FANCD2, and RAD52. ATR and FANCD2 are interdependently recruited. The DDR may promote completion of replication, because SLX4 and ATR inhibition exacerbates mitotic abnormality induced by LacI. The underreplicated intermediates persisting until late G2/M phase are processed by MUS81-mediated cleavage in early mitosis. Note that cleaved strands are arbitrary in the figure, because it remains unclear whether the cleavage occurs in the leading or lagging strand templates. Cleavage at lacO can promote MiDAS (∼20% in the absence or presence of LacI; ) or end joining that results in deletion of the loci (∼30% in the absence of LacI and ∼50% in the presence of LacI; ). A fraction of the lacO arrays escape from the cleavage and remain underreplicated until anaphase, leading to the anaphase abnormal lacO structure (∼5% in the absence of LacI and ∼20% in the presence of LacI; ) and the formation of 53BP1 NBs in daughter G1 cells (∼5% in the absence of LacI and ∼20% in the presence of LacI; ).

    Techniques Used: Binding Assay, Generated, Inhibition

    LacI binding in S phase promotes a UFB-like anaphase abnormal lacO array and underreplicated DNA lesions in daughter cells. (A and B) U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase by treatment with 7 µM RO-3306 for 20 h and 1 µM 4-OHT (for LacI induction) for 20 h or the last 4 h. The G2-arrested cells were then released into fresh medium for 45 min to enable progression into anaphase and immunostained with the indicated antibodies, followed by DAPI staining and analysis. (A) Representative images of anaphase cells. Yellow arrows indicate DAPI-negative anaphase abnormal lacO arrays colocalized with RPA and PICH. White arrows denote normally segregated lacO arrays. Scale bars, 10 µm. (B) The graph indicates frequencies of anaphase abnormal lacO per anaphase cell. Values are sum scores from two independent experiments. ***, P < 0.001 (χ 2 test). Individual data points from the two independent experiments are also shown. (C–E) U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase and released into G1 phase as described in . Cells were double immunostained with anti-LacI and anti-53BP1 antibodies, followed by DAPI staining and analysis. (C) Representative images of twin 53BP1 NBs (indicated by arrows) that colocalize with the symmetrical LacI foci in the sister cells. Scale bar, 10 µm. (D) A diagram depicting different patterns of 53BP1 NBs in the sister cells with symmetrical LacI foci. (E) The graph indicates frequencies of sister pairs with twin 53BP1 NBs as a percentage of sister pairs harboring symmetrical LacI foci. Values are sum scores from two independent experiments. **, P < 0.01 (χ 2 test). Individual data points from the two independent experiments are also shown.
    Figure Legend Snippet: LacI binding in S phase promotes a UFB-like anaphase abnormal lacO array and underreplicated DNA lesions in daughter cells. (A and B) U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase by treatment with 7 µM RO-3306 for 20 h and 1 µM 4-OHT (for LacI induction) for 20 h or the last 4 h. The G2-arrested cells were then released into fresh medium for 45 min to enable progression into anaphase and immunostained with the indicated antibodies, followed by DAPI staining and analysis. (A) Representative images of anaphase cells. Yellow arrows indicate DAPI-negative anaphase abnormal lacO arrays colocalized with RPA and PICH. White arrows denote normally segregated lacO arrays. Scale bars, 10 µm. (B) The graph indicates frequencies of anaphase abnormal lacO per anaphase cell. Values are sum scores from two independent experiments. ***, P < 0.001 (χ 2 test). Individual data points from the two independent experiments are also shown. (C–E) U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase and released into G1 phase as described in . Cells were double immunostained with anti-LacI and anti-53BP1 antibodies, followed by DAPI staining and analysis. (C) Representative images of twin 53BP1 NBs (indicated by arrows) that colocalize with the symmetrical LacI foci in the sister cells. Scale bar, 10 µm. (D) A diagram depicting different patterns of 53BP1 NBs in the sister cells with symmetrical LacI foci. (E) The graph indicates frequencies of sister pairs with twin 53BP1 NBs as a percentage of sister pairs harboring symmetrical LacI foci. Values are sum scores from two independent experiments. **, P < 0.01 (χ 2 test). Individual data points from the two independent experiments are also shown.

    Techniques Used: Binding Assay, Staining

    53bp1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 53bp1
    Recruitment of various DDR proteins to lacO arrays in U2OS 40–2-6 ER-LacI cells. (A) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT for 2 h were double immunostained with the indicated antibodies and counterstained with DAPI. ssDNA was stained with anti-BrdU antibody under nondenaturing conditions. For detection of total ssDNA, DNA was labeled for 24 h with 10 µM BrdU. For detection of nascent-strand ssDNA, newly synthesized DNA was labeled with BrdU during treatment with 4-OHT. For detection of parental-strand ssDNA, DNA was labeled with 10 µM BrdU for 20 h, followed by a chase in fresh medium without BrdU for 2 h before addition of 4-OHT. Representative images are shown, and the colocalization frequencies are shown in . Yellow arrows indicate colocalization of DDR proteins with the foci of HA-LacI, and white arrows indicate noncolocalization. Scale bars, 10 µm. (B) The antibody used in this study can detect bleomycin-induced RAD51 foci. U2OS 40–2-6 ER-LacI cells were treated with 8 µM Bleomycin for 18 h, then immunostained with anti-RAD51 antibody, followed by DAPI staining. Representative images are shown. Scale bar, 10 µm. (C) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT or a control vehicle (EtOH) for 2 h were subjected to ChIP-quantitative PCR analysis using the indicated antibodies. Enrichment of the lacO sequences is shown as the percent of input DNA. Means ± SD are shown ( n = 6 for control rabbit IgG; n = 7 for LacI and <t>53BP1).</t> *, P < 0.05; ***, P < 0.001; n.s., not significant (two-tailed Student’s t test).
    53bp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/53bp1/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    53bp1 - by Bioz Stars, 2023-01
    93/100 stars

    Images

    1) Product Images from "SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions"

    Article Title: SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.202003148

    Recruitment of various DDR proteins to lacO arrays in U2OS 40–2-6 ER-LacI cells. (A) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT for 2 h were double immunostained with the indicated antibodies and counterstained with DAPI. ssDNA was stained with anti-BrdU antibody under nondenaturing conditions. For detection of total ssDNA, DNA was labeled for 24 h with 10 µM BrdU. For detection of nascent-strand ssDNA, newly synthesized DNA was labeled with BrdU during treatment with 4-OHT. For detection of parental-strand ssDNA, DNA was labeled with 10 µM BrdU for 20 h, followed by a chase in fresh medium without BrdU for 2 h before addition of 4-OHT. Representative images are shown, and the colocalization frequencies are shown in . Yellow arrows indicate colocalization of DDR proteins with the foci of HA-LacI, and white arrows indicate noncolocalization. Scale bars, 10 µm. (B) The antibody used in this study can detect bleomycin-induced RAD51 foci. U2OS 40–2-6 ER-LacI cells were treated with 8 µM Bleomycin for 18 h, then immunostained with anti-RAD51 antibody, followed by DAPI staining. Representative images are shown. Scale bar, 10 µm. (C) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT or a control vehicle (EtOH) for 2 h were subjected to ChIP-quantitative PCR analysis using the indicated antibodies. Enrichment of the lacO sequences is shown as the percent of input DNA. Means ± SD are shown ( n = 6 for control rabbit IgG; n = 7 for LacI and 53BP1). *, P < 0.05; ***, P < 0.001; n.s., not significant (two-tailed Student’s t test).
    Figure Legend Snippet: Recruitment of various DDR proteins to lacO arrays in U2OS 40–2-6 ER-LacI cells. (A) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT for 2 h were double immunostained with the indicated antibodies and counterstained with DAPI. ssDNA was stained with anti-BrdU antibody under nondenaturing conditions. For detection of total ssDNA, DNA was labeled for 24 h with 10 µM BrdU. For detection of nascent-strand ssDNA, newly synthesized DNA was labeled with BrdU during treatment with 4-OHT. For detection of parental-strand ssDNA, DNA was labeled with 10 µM BrdU for 20 h, followed by a chase in fresh medium without BrdU for 2 h before addition of 4-OHT. Representative images are shown, and the colocalization frequencies are shown in . Yellow arrows indicate colocalization of DDR proteins with the foci of HA-LacI, and white arrows indicate noncolocalization. Scale bars, 10 µm. (B) The antibody used in this study can detect bleomycin-induced RAD51 foci. U2OS 40–2-6 ER-LacI cells were treated with 8 µM Bleomycin for 18 h, then immunostained with anti-RAD51 antibody, followed by DAPI staining. Representative images are shown. Scale bar, 10 µm. (C) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT or a control vehicle (EtOH) for 2 h were subjected to ChIP-quantitative PCR analysis using the indicated antibodies. Enrichment of the lacO sequences is shown as the percent of input DNA. Means ± SD are shown ( n = 6 for control rabbit IgG; n = 7 for LacI and 53BP1). *, P < 0.05; ***, P < 0.001; n.s., not significant (two-tailed Student’s t test).

    Techniques Used: Staining, Labeling, Synthesized, Real-time Polymerase Chain Reaction, Two Tailed Test

    A model of the fate of underreplicated DNA regions in the lacO array. Even in the absence of LacI binding, the lacO array is a difficult-to-replicate region, like other repeat sequences, and contains underreplicated lesions (∼60% on the basis of the data shown in ), which persist until late G2/M phase. Replication stress by LacI binding to the array further increases the frequency of cells with underreplicated DNA lesions (∼90%). To try to complete replication of the array during S phase, cells activate the DDR, in which one-ended DSB is first generated by SLX4–XPF–mediated DNA cleavage. Excess ssDNA is then generated by unidentified exonucleases, which in turn recruit ATR, FANCD2, and RAD52. ATR and FANCD2 are interdependently recruited. The DDR may promote completion of replication, because SLX4 and ATR inhibition exacerbates mitotic abnormality induced by LacI. The underreplicated intermediates persisting until late G2/M phase are processed by MUS81-mediated cleavage in early mitosis. Note that cleaved strands are arbitrary in the figure, because it remains unclear whether the cleavage occurs in the leading or lagging strand templates. Cleavage at lacO can promote MiDAS (∼20% in the absence or presence of LacI; ) or end joining that results in deletion of the loci (∼30% in the absence of LacI and ∼50% in the presence of LacI; ). A fraction of the lacO arrays escape from the cleavage and remain underreplicated until anaphase, leading to the anaphase abnormal lacO structure (∼5% in the absence of LacI and ∼20% in the presence of LacI; ) and the formation of 53BP1 NBs in daughter G1 cells (∼5% in the absence of LacI and ∼20% in the presence of LacI; ).
    Figure Legend Snippet: A model of the fate of underreplicated DNA regions in the lacO array. Even in the absence of LacI binding, the lacO array is a difficult-to-replicate region, like other repeat sequences, and contains underreplicated lesions (∼60% on the basis of the data shown in ), which persist until late G2/M phase. Replication stress by LacI binding to the array further increases the frequency of cells with underreplicated DNA lesions (∼90%). To try to complete replication of the array during S phase, cells activate the DDR, in which one-ended DSB is first generated by SLX4–XPF–mediated DNA cleavage. Excess ssDNA is then generated by unidentified exonucleases, which in turn recruit ATR, FANCD2, and RAD52. ATR and FANCD2 are interdependently recruited. The DDR may promote completion of replication, because SLX4 and ATR inhibition exacerbates mitotic abnormality induced by LacI. The underreplicated intermediates persisting until late G2/M phase are processed by MUS81-mediated cleavage in early mitosis. Note that cleaved strands are arbitrary in the figure, because it remains unclear whether the cleavage occurs in the leading or lagging strand templates. Cleavage at lacO can promote MiDAS (∼20% in the absence or presence of LacI; ) or end joining that results in deletion of the loci (∼30% in the absence of LacI and ∼50% in the presence of LacI; ). A fraction of the lacO arrays escape from the cleavage and remain underreplicated until anaphase, leading to the anaphase abnormal lacO structure (∼5% in the absence of LacI and ∼20% in the presence of LacI; ) and the formation of 53BP1 NBs in daughter G1 cells (∼5% in the absence of LacI and ∼20% in the presence of LacI; ).

    Techniques Used: Binding Assay, Generated, Inhibition

    LacI binding in S phase promotes a UFB-like anaphase abnormal lacO array and underreplicated DNA lesions in daughter cells. (A and B) U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase by treatment with 7 µM RO-3306 for 20 h and 1 µM 4-OHT (for LacI induction) for 20 h or the last 4 h. The G2-arrested cells were then released into fresh medium for 45 min to enable progression into anaphase and immunostained with the indicated antibodies, followed by DAPI staining and analysis. (A) Representative images of anaphase cells. Yellow arrows indicate DAPI-negative anaphase abnormal lacO arrays colocalized with RPA and PICH. White arrows denote normally segregated lacO arrays. Scale bars, 10 µm. (B) The graph indicates frequencies of anaphase abnormal lacO per anaphase cell. Values are sum scores from two independent experiments. ***, P < 0.001 (χ 2 test). Individual data points from the two independent experiments are also shown. (C–E) U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase and released into G1 phase as described in . Cells were double immunostained with anti-LacI and anti-53BP1 antibodies, followed by DAPI staining and analysis. (C) Representative images of twin 53BP1 NBs (indicated by arrows) that colocalize with the symmetrical LacI foci in the sister cells. Scale bar, 10 µm. (D) A diagram depicting different patterns of 53BP1 NBs in the sister cells with symmetrical LacI foci. (E) The graph indicates frequencies of sister pairs with twin 53BP1 NBs as a percentage of sister pairs harboring symmetrical LacI foci. Values are sum scores from two independent experiments. **, P < 0.01 (χ 2 test). Individual data points from the two independent experiments are also shown.
    Figure Legend Snippet: LacI binding in S phase promotes a UFB-like anaphase abnormal lacO array and underreplicated DNA lesions in daughter cells. (A and B) U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase by treatment with 7 µM RO-3306 for 20 h and 1 µM 4-OHT (for LacI induction) for 20 h or the last 4 h. The G2-arrested cells were then released into fresh medium for 45 min to enable progression into anaphase and immunostained with the indicated antibodies, followed by DAPI staining and analysis. (A) Representative images of anaphase cells. Yellow arrows indicate DAPI-negative anaphase abnormal lacO arrays colocalized with RPA and PICH. White arrows denote normally segregated lacO arrays. Scale bars, 10 µm. (B) The graph indicates frequencies of anaphase abnormal lacO per anaphase cell. Values are sum scores from two independent experiments. ***, P < 0.001 (χ 2 test). Individual data points from the two independent experiments are also shown. (C–E) U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase and released into G1 phase as described in . Cells were double immunostained with anti-LacI and anti-53BP1 antibodies, followed by DAPI staining and analysis. (C) Representative images of twin 53BP1 NBs (indicated by arrows) that colocalize with the symmetrical LacI foci in the sister cells. Scale bar, 10 µm. (D) A diagram depicting different patterns of 53BP1 NBs in the sister cells with symmetrical LacI foci. (E) The graph indicates frequencies of sister pairs with twin 53BP1 NBs as a percentage of sister pairs harboring symmetrical LacI foci. Values are sum scores from two independent experiments. **, P < 0.01 (χ 2 test). Individual data points from the two independent experiments are also shown.

    Techniques Used: Binding Assay, Staining

    53bp1  (Cell Signaling Technology Inc)


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    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc 53bp1
    A Schematic diagram of establishment of KLF4 loxp/loxp coupled AAV7‐Cre inducible mouse. B Genotyping of KLF4 loxp/loxp coupled AAV‐Cre inducible mouse. 2 × 10 11 particles of AAV7‐Cre‐mCherry or AAV7‐mCherry were intraperitoneally injected into KLF4 loxp/loxp mice (6–8 weeks). Five weeks later, mice tails were cut and collected for DNA extraction and PCR analysis. PCR results were analyzed by 1% agarose gel. C, D Validation of inducible knockout of KLF4 in mouse intestine. Five weeks after injection of AAV7‐Cre‐mCherry, mouse intestine was removed and followed by the preparation of tissue section. The KLF4 expression in the intestine was then detected by Western blot (C) and immunohistochemistry (D). Scale bars, 100 μm. E Kaplan–Meier survival curves of KLF4 loxp/loxp mice with intraperitoneal injection of AAV7‐Cre‐mCherry or AAV7‐mCherry followed by the treatment with 8‐Gy (total‐body) γ‐irradiation 5 weeks after then. AAV7‐mCherry, n = 10 per group; AAV7‐Cre‐mCherry, n = 12 per group. P = 0.0023, log‐rank test. F Histological analysis of intestinal epithelium of KLF4 loxp/loxp mice with AAV7‐mCherry or AAV7‐Cre‐mCherry intraperitoneal injection followed by 8‐Gy (total‐body) γ‐irradiation 5 weeks after then. Tissues were collected from the sham mice and mice at different time after exposure to γ‐irradiation. Scale bars, 100 μm. G, H Immunofluorescent staining of γ‐H2AX, <t>53BP1,</t> and cleaved caspase‐3 in the intestinal epithelium of KLF4 loxp/loxp mice with injection of AAV7‐mCherry or AAV7‐Cre‐mCherry followed by treatment of γ‐irradiation. Tissues were collected from sham mice and mice at different time after exposure to γ‐irradiation and then staining with indicated antibodies. (G) Quantification of γ‐H2AX, <t>53BP1</t> and active caspase‐3‐positive cells based on the Immunofluorescent staining results presented in (H). Data are mean ± SEM; n = 4 per group; P = 0.0023 (r‐H2AX), P = 7.9 × 10 −4 (53BP1) P = 5.5 × 10 −3 (active caspase‐3). One‐way ANOVA was used for the statistical analysis. Scale bars, 60 μm.
    53bp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/53bp1/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    53bp1 - by Bioz Stars, 2023-01
    93/100 stars

    Images

    1) Product Images from "New insight into the significance of KLF4 PARylation in genome stability, carcinogenesis, and therapy"

    Article Title: New insight into the significance of KLF4 PARylation in genome stability, carcinogenesis, and therapy

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.202012391

    A Schematic diagram of establishment of KLF4 loxp/loxp coupled AAV7‐Cre inducible mouse. B Genotyping of KLF4 loxp/loxp coupled AAV‐Cre inducible mouse. 2 × 10 11 particles of AAV7‐Cre‐mCherry or AAV7‐mCherry were intraperitoneally injected into KLF4 loxp/loxp mice (6–8 weeks). Five weeks later, mice tails were cut and collected for DNA extraction and PCR analysis. PCR results were analyzed by 1% agarose gel. C, D Validation of inducible knockout of KLF4 in mouse intestine. Five weeks after injection of AAV7‐Cre‐mCherry, mouse intestine was removed and followed by the preparation of tissue section. The KLF4 expression in the intestine was then detected by Western blot (C) and immunohistochemistry (D). Scale bars, 100 μm. E Kaplan–Meier survival curves of KLF4 loxp/loxp mice with intraperitoneal injection of AAV7‐Cre‐mCherry or AAV7‐mCherry followed by the treatment with 8‐Gy (total‐body) γ‐irradiation 5 weeks after then. AAV7‐mCherry, n = 10 per group; AAV7‐Cre‐mCherry, n = 12 per group. P = 0.0023, log‐rank test. F Histological analysis of intestinal epithelium of KLF4 loxp/loxp mice with AAV7‐mCherry or AAV7‐Cre‐mCherry intraperitoneal injection followed by 8‐Gy (total‐body) γ‐irradiation 5 weeks after then. Tissues were collected from the sham mice and mice at different time after exposure to γ‐irradiation. Scale bars, 100 μm. G, H Immunofluorescent staining of γ‐H2AX, 53BP1, and cleaved caspase‐3 in the intestinal epithelium of KLF4 loxp/loxp mice with injection of AAV7‐mCherry or AAV7‐Cre‐mCherry followed by treatment of γ‐irradiation. Tissues were collected from sham mice and mice at different time after exposure to γ‐irradiation and then staining with indicated antibodies. (G) Quantification of γ‐H2AX, 53BP1 and active caspase‐3‐positive cells based on the Immunofluorescent staining results presented in (H). Data are mean ± SEM; n = 4 per group; P = 0.0023 (r‐H2AX), P = 7.9 × 10 −4 (53BP1) P = 5.5 × 10 −3 (active caspase‐3). One‐way ANOVA was used for the statistical analysis. Scale bars, 60 μm.
    Figure Legend Snippet: A Schematic diagram of establishment of KLF4 loxp/loxp coupled AAV7‐Cre inducible mouse. B Genotyping of KLF4 loxp/loxp coupled AAV‐Cre inducible mouse. 2 × 10 11 particles of AAV7‐Cre‐mCherry or AAV7‐mCherry were intraperitoneally injected into KLF4 loxp/loxp mice (6–8 weeks). Five weeks later, mice tails were cut and collected for DNA extraction and PCR analysis. PCR results were analyzed by 1% agarose gel. C, D Validation of inducible knockout of KLF4 in mouse intestine. Five weeks after injection of AAV7‐Cre‐mCherry, mouse intestine was removed and followed by the preparation of tissue section. The KLF4 expression in the intestine was then detected by Western blot (C) and immunohistochemistry (D). Scale bars, 100 μm. E Kaplan–Meier survival curves of KLF4 loxp/loxp mice with intraperitoneal injection of AAV7‐Cre‐mCherry or AAV7‐mCherry followed by the treatment with 8‐Gy (total‐body) γ‐irradiation 5 weeks after then. AAV7‐mCherry, n = 10 per group; AAV7‐Cre‐mCherry, n = 12 per group. P = 0.0023, log‐rank test. F Histological analysis of intestinal epithelium of KLF4 loxp/loxp mice with AAV7‐mCherry or AAV7‐Cre‐mCherry intraperitoneal injection followed by 8‐Gy (total‐body) γ‐irradiation 5 weeks after then. Tissues were collected from the sham mice and mice at different time after exposure to γ‐irradiation. Scale bars, 100 μm. G, H Immunofluorescent staining of γ‐H2AX, 53BP1, and cleaved caspase‐3 in the intestinal epithelium of KLF4 loxp/loxp mice with injection of AAV7‐mCherry or AAV7‐Cre‐mCherry followed by treatment of γ‐irradiation. Tissues were collected from sham mice and mice at different time after exposure to γ‐irradiation and then staining with indicated antibodies. (G) Quantification of γ‐H2AX, 53BP1 and active caspase‐3‐positive cells based on the Immunofluorescent staining results presented in (H). Data are mean ± SEM; n = 4 per group; P = 0.0023 (r‐H2AX), P = 7.9 × 10 −4 (53BP1) P = 5.5 × 10 −3 (active caspase‐3). One‐way ANOVA was used for the statistical analysis. Scale bars, 60 μm.

    Techniques Used: Injection, DNA Extraction, Agarose Gel Electrophoresis, Knock-Out, Expressing, Western Blot, Immunohistochemistry, Irradiation, Staining

    A Depletion of KLF4 directly diminishes the DNA damage‐induced p21 expression, resulting in the failure of cell cycle arrest in MDA‐MB‐231 cells. B Abolishment of KLF4 PARylation disrupts KLF4‐mediated p21 expression that in turn impairs DNA damage response in MDA‐MB‐231 cells. C p‐H3 staining analysis of KLF4 +/+ , KLF4 −/− MEFs, KLF4 −/− MEF with transfection of wild‐type or KLF4‐Zinc 2‐YYR/AAA mutation. n = 4, P values were labeled in figure, one‐way ANOVA assay. D, E Abolishment of KLF4 PARylation leads to failure in removing damaged DNA as measured by 53BP1 foci. n = 4, P = 4.74 × 10 −6 (KLF4 WT vs. KLF4 YYR/AAA ), one‐way ANOVA assay. (E) Summary of (D). F Failure of KLF4 PARylation leads to increased aneuploidy population in U2OS cells. n = 3, P values were labeled in figure, one‐way ANOVA assay. G Effect of KLF4 PARylation on NHEJ and HR. Wild‐type or KLF4‐Zinc 2‐YYR/AAA mutant KLF4 were co‐transfected with I‐SCE construction in U2Os‐GFP‐EJ5 cells (for NHEJ assay) or U2Os‐DR‐GFP (for HR assay). n = 3, P values were labeled in figure, one‐way ANOVA assay. H–J The effect of overexpression or knockdown KLF4 on mRNA levels of BRCA1 in KLF4 −/− MEF (H), MDA‐MB‐468 (I) and MDA‐MB‐231 (J). n = 3, P values were labeled in figures, one‐way ANOVA assay. K Heatmap of BRCA1, P21, Bax expression on U2OS cells with expression of wild‐type KLF4 or KLF4‐Zinc 2‐YYR/AAA mutant as well as KLF4‐Zinc 2 (Zinc 2 domain deletion) mutant. No difference of BRCA1 expression was measured between expression of wild‐type KLF4 or KLF4‐Zinc 2‐YYR/AAA mutant, while loss of Zinc domain causes drops of BRCA1 expression levels. L In early responsive window (1–2 h after exposure to γ‐radiation), while elevation of wild‐type KLF4 enhances the expression levels of p21, disruption of KLF4 PARylation diminishes KLF4‐mediated p21 accumulation. Upon DNA damage, no matter PARylation or not, elevation of both wild‐type or KLF4‐PARylation‐deficient mutant leads to BRCA1 accumulation suggesting the KLF4‐mediated regulation of BRCA1 is independent of PARP1 in MDA‐MB‐231 cells. M KLF4 physically interacts with BRCA1 upstream promoter region measured by CHIP‐PCR. N inset, top, schematic diagram of the BRCA1 promoter and relative positions of primer sets used in this study. ChIP analysis at the BRCA1 promoter using KLF4‐specific or nonspecific control IgG (α‐Gal4) in MDA‐MB‐231 cells. Shown is the enrichment at positions of the BRCA1 locus relative to the TSS, presented as percent recovery of input. O inset, top, schematic diagram of the BRCA1 promoter cloning primer and the alignment of potential KLF4 binding motif on BRCA1 promoter with KLF4 binding motif on p21 and SLC5A6 promoter. Shown is the wild‐type (BRCA1‐WT) or mutant (BRCA1‐AA) BRCA1 promoter luciferase reporter activity when co‐transfect with KLF4 plasmids. KLF4 co‐transfection promotes BRCA1‐WT but not BRCA1‐AA promoter reporter transcription. n = 3, P = 0.018, one‐way ANOVA assay. P ChIP analysis of KLF4 binding to the BRCA1 promoter in MDA‐MB‐231 at −0.3K positions relative to the TSS in untreated and olaparib (10 µM for 8 h) or 5 Gy radiation treat cells. No significant difference of KLF4 binds to BRCA1 promoter between untreated and olaparib or radiation treat cells. n = 3, P = 0.80, one‐way ANOVA assay. Q BRCA1 reporter assay in KLF4 −/− MEFs, KLF4 −/− MEF with transfection of wild‐type (KLF4 WT ) or mutant KLF4 (KLF4 YYR/AAA , KLF4 C403A and KLF4 ΔZinc2 ). The depleted the zinc domain or mutated zinc finger (KLF4 C403A and KLF4 ΔZinc2 ) on KLF4 impairs the KLF4‐driven BRCA1 expression, while no effect was observed between wild‐type and KLF4 YYR/AAA mutant. n = 3, P values were labeled in figure, one‐way ANOVA assay. R HR analysis. U2OS‐DR‐GFP cells were transfected with I‐SceI, BRCA1, and siBRCA1 in KLF4‐wild‐type and depletion condition, respectively. GFP‐positive cells representing HR repair rate were measured by flow cytometry 48–72 h after then. Overexpression of BRCA1 restores the HR efficiency in KLF4 knockdown cells. Data information: Data are mean ± SEM; n = 3, P values were labeled in figure, one‐way ANOVA assay.
    Figure Legend Snippet: A Depletion of KLF4 directly diminishes the DNA damage‐induced p21 expression, resulting in the failure of cell cycle arrest in MDA‐MB‐231 cells. B Abolishment of KLF4 PARylation disrupts KLF4‐mediated p21 expression that in turn impairs DNA damage response in MDA‐MB‐231 cells. C p‐H3 staining analysis of KLF4 +/+ , KLF4 −/− MEFs, KLF4 −/− MEF with transfection of wild‐type or KLF4‐Zinc 2‐YYR/AAA mutation. n = 4, P values were labeled in figure, one‐way ANOVA assay. D, E Abolishment of KLF4 PARylation leads to failure in removing damaged DNA as measured by 53BP1 foci. n = 4, P = 4.74 × 10 −6 (KLF4 WT vs. KLF4 YYR/AAA ), one‐way ANOVA assay. (E) Summary of (D). F Failure of KLF4 PARylation leads to increased aneuploidy population in U2OS cells. n = 3, P values were labeled in figure, one‐way ANOVA assay. G Effect of KLF4 PARylation on NHEJ and HR. Wild‐type or KLF4‐Zinc 2‐YYR/AAA mutant KLF4 were co‐transfected with I‐SCE construction in U2Os‐GFP‐EJ5 cells (for NHEJ assay) or U2Os‐DR‐GFP (for HR assay). n = 3, P values were labeled in figure, one‐way ANOVA assay. H–J The effect of overexpression or knockdown KLF4 on mRNA levels of BRCA1 in KLF4 −/− MEF (H), MDA‐MB‐468 (I) and MDA‐MB‐231 (J). n = 3, P values were labeled in figures, one‐way ANOVA assay. K Heatmap of BRCA1, P21, Bax expression on U2OS cells with expression of wild‐type KLF4 or KLF4‐Zinc 2‐YYR/AAA mutant as well as KLF4‐Zinc 2 (Zinc 2 domain deletion) mutant. No difference of BRCA1 expression was measured between expression of wild‐type KLF4 or KLF4‐Zinc 2‐YYR/AAA mutant, while loss of Zinc domain causes drops of BRCA1 expression levels. L In early responsive window (1–2 h after exposure to γ‐radiation), while elevation of wild‐type KLF4 enhances the expression levels of p21, disruption of KLF4 PARylation diminishes KLF4‐mediated p21 accumulation. Upon DNA damage, no matter PARylation or not, elevation of both wild‐type or KLF4‐PARylation‐deficient mutant leads to BRCA1 accumulation suggesting the KLF4‐mediated regulation of BRCA1 is independent of PARP1 in MDA‐MB‐231 cells. M KLF4 physically interacts with BRCA1 upstream promoter region measured by CHIP‐PCR. N inset, top, schematic diagram of the BRCA1 promoter and relative positions of primer sets used in this study. ChIP analysis at the BRCA1 promoter using KLF4‐specific or nonspecific control IgG (α‐Gal4) in MDA‐MB‐231 cells. Shown is the enrichment at positions of the BRCA1 locus relative to the TSS, presented as percent recovery of input. O inset, top, schematic diagram of the BRCA1 promoter cloning primer and the alignment of potential KLF4 binding motif on BRCA1 promoter with KLF4 binding motif on p21 and SLC5A6 promoter. Shown is the wild‐type (BRCA1‐WT) or mutant (BRCA1‐AA) BRCA1 promoter luciferase reporter activity when co‐transfect with KLF4 plasmids. KLF4 co‐transfection promotes BRCA1‐WT but not BRCA1‐AA promoter reporter transcription. n = 3, P = 0.018, one‐way ANOVA assay. P ChIP analysis of KLF4 binding to the BRCA1 promoter in MDA‐MB‐231 at −0.3K positions relative to the TSS in untreated and olaparib (10 µM for 8 h) or 5 Gy radiation treat cells. No significant difference of KLF4 binds to BRCA1 promoter between untreated and olaparib or radiation treat cells. n = 3, P = 0.80, one‐way ANOVA assay. Q BRCA1 reporter assay in KLF4 −/− MEFs, KLF4 −/− MEF with transfection of wild‐type (KLF4 WT ) or mutant KLF4 (KLF4 YYR/AAA , KLF4 C403A and KLF4 ΔZinc2 ). The depleted the zinc domain or mutated zinc finger (KLF4 C403A and KLF4 ΔZinc2 ) on KLF4 impairs the KLF4‐driven BRCA1 expression, while no effect was observed between wild‐type and KLF4 YYR/AAA mutant. n = 3, P values were labeled in figure, one‐way ANOVA assay. R HR analysis. U2OS‐DR‐GFP cells were transfected with I‐SceI, BRCA1, and siBRCA1 in KLF4‐wild‐type and depletion condition, respectively. GFP‐positive cells representing HR repair rate were measured by flow cytometry 48–72 h after then. Overexpression of BRCA1 restores the HR efficiency in KLF4 knockdown cells. Data information: Data are mean ± SEM; n = 3, P values were labeled in figure, one‐way ANOVA assay.

    Techniques Used: Expressing, Staining, Transfection, Mutagenesis, Labeling, NHEJ Assay, Over Expression, Clone Assay, Binding Assay, Luciferase, Activity Assay, Cotransfection, Reporter Assay, Flow Cytometry

    53bp1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 53bp1
    The RAD18–SLF1 interaction promotes DNA fiber progression. ( A ) MDA-MB-231 cells were transduced to express ectopic RAD18 or GFP and treated with scrambled siRNA or siRNA targeting the 3′ UTR of the endogenous RAD18. Lysates were subject to western blotting to confirm knockdown of endogenous RAD18 and reconstitution with ectopic RAD18 ( left ). MDA-MB-231 cells with ectopic RAD18 in combinations with dox-induced RNF168 and scrambled or BRCA1 targeted siRNA were incubated with CldU for 40 min followed by a 24 h co-incubation of HU and IdU. DNA fiber lengths were assessed and are presented as the IdU/CldU length ratio, black bar indicates median values (median numbers are also shown below). A minimum of 120 replication forks were measured for each condition. *** P < 0.001 (Mann–Whitney test) compared to the scrambled siRNA treated control. ( B ) The indicated RAD18 cDNA constructs and mutations were generated. RAD18 constructs and a GFP control were expressed in dox-inducible RNF168 MDA-MB-231 cells and assessed for expression by western blotting. ( C ) Cell lines from (B) were treated with siRNA targeting BRCA1 and RAD18 3′ UTR and fibers assessed as described in (A). A minimum of 150 replication forks were measured for each cell line. *** P < 0.001 (Mann–Whitney test) compared to the GFP expressing control. ( D ) Dox-inducible RNF168 expressing MDA-MB-231 cells were treated with the indicated siRNAs and fibers assessed as described in (A). A minimum of 200 replication forks were measured for each condition. *** P < 0.001 (Mann–Whitney test). ( E ) Dox-inducible GFP or RNF168 expressing MDA-MB-231 cells were treated with 4 mM HU or vehicle for 24 h and subjected to western blotting for the indicated proteins. ( F ) One-ended DSBs arising at HU-induced stalled replication forks activate RNF168, which subsequently localizes <t>53BP1</t> and RAD18 to ub-H2AX. In the presence of BRCA1, HR repair ensues. In the absence of BRCA1, 53BP1 inhibits DNA end resection while RAD18 promotes BIR.
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    1) Product Images from "Ectopic RNF168 expression promotes break-induced replication-like DNA synthesis at stalled replication forks"

    Article Title: Ectopic RNF168 expression promotes break-induced replication-like DNA synthesis at stalled replication forks

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa154

    The RAD18–SLF1 interaction promotes DNA fiber progression. ( A ) MDA-MB-231 cells were transduced to express ectopic RAD18 or GFP and treated with scrambled siRNA or siRNA targeting the 3′ UTR of the endogenous RAD18. Lysates were subject to western blotting to confirm knockdown of endogenous RAD18 and reconstitution with ectopic RAD18 ( left ). MDA-MB-231 cells with ectopic RAD18 in combinations with dox-induced RNF168 and scrambled or BRCA1 targeted siRNA were incubated with CldU for 40 min followed by a 24 h co-incubation of HU and IdU. DNA fiber lengths were assessed and are presented as the IdU/CldU length ratio, black bar indicates median values (median numbers are also shown below). A minimum of 120 replication forks were measured for each condition. *** P < 0.001 (Mann–Whitney test) compared to the scrambled siRNA treated control. ( B ) The indicated RAD18 cDNA constructs and mutations were generated. RAD18 constructs and a GFP control were expressed in dox-inducible RNF168 MDA-MB-231 cells and assessed for expression by western blotting. ( C ) Cell lines from (B) were treated with siRNA targeting BRCA1 and RAD18 3′ UTR and fibers assessed as described in (A). A minimum of 150 replication forks were measured for each cell line. *** P < 0.001 (Mann–Whitney test) compared to the GFP expressing control. ( D ) Dox-inducible RNF168 expressing MDA-MB-231 cells were treated with the indicated siRNAs and fibers assessed as described in (A). A minimum of 200 replication forks were measured for each condition. *** P < 0.001 (Mann–Whitney test). ( E ) Dox-inducible GFP or RNF168 expressing MDA-MB-231 cells were treated with 4 mM HU or vehicle for 24 h and subjected to western blotting for the indicated proteins. ( F ) One-ended DSBs arising at HU-induced stalled replication forks activate RNF168, which subsequently localizes 53BP1 and RAD18 to ub-H2AX. In the presence of BRCA1, HR repair ensues. In the absence of BRCA1, 53BP1 inhibits DNA end resection while RAD18 promotes BIR.
    Figure Legend Snippet: The RAD18–SLF1 interaction promotes DNA fiber progression. ( A ) MDA-MB-231 cells were transduced to express ectopic RAD18 or GFP and treated with scrambled siRNA or siRNA targeting the 3′ UTR of the endogenous RAD18. Lysates were subject to western blotting to confirm knockdown of endogenous RAD18 and reconstitution with ectopic RAD18 ( left ). MDA-MB-231 cells with ectopic RAD18 in combinations with dox-induced RNF168 and scrambled or BRCA1 targeted siRNA were incubated with CldU for 40 min followed by a 24 h co-incubation of HU and IdU. DNA fiber lengths were assessed and are presented as the IdU/CldU length ratio, black bar indicates median values (median numbers are also shown below). A minimum of 120 replication forks were measured for each condition. *** P < 0.001 (Mann–Whitney test) compared to the scrambled siRNA treated control. ( B ) The indicated RAD18 cDNA constructs and mutations were generated. RAD18 constructs and a GFP control were expressed in dox-inducible RNF168 MDA-MB-231 cells and assessed for expression by western blotting. ( C ) Cell lines from (B) were treated with siRNA targeting BRCA1 and RAD18 3′ UTR and fibers assessed as described in (A). A minimum of 150 replication forks were measured for each cell line. *** P < 0.001 (Mann–Whitney test) compared to the GFP expressing control. ( D ) Dox-inducible RNF168 expressing MDA-MB-231 cells were treated with the indicated siRNAs and fibers assessed as described in (A). A minimum of 200 replication forks were measured for each condition. *** P < 0.001 (Mann–Whitney test). ( E ) Dox-inducible GFP or RNF168 expressing MDA-MB-231 cells were treated with 4 mM HU or vehicle for 24 h and subjected to western blotting for the indicated proteins. ( F ) One-ended DSBs arising at HU-induced stalled replication forks activate RNF168, which subsequently localizes 53BP1 and RAD18 to ub-H2AX. In the presence of BRCA1, HR repair ensues. In the absence of BRCA1, 53BP1 inhibits DNA end resection while RAD18 promotes BIR.

    Techniques Used: Western Blot, Incubation, MANN-WHITNEY, Construct, Generated, Expressing

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    Cell Signaling Technology Inc 53bp1
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    Cell Signaling Technology Inc α 53bp1 antibody
    Increased MH usage in Sμ−Sγ1 and Sμ−Sε CSR junctions from DSBR factor-deficient primary B cells. (A) Illustration of joining outcomes of Sμ region DSBs to downstream Sγ1 and Sε region breaks undergoing direct or MH-mediated joining. WT, ATM−/,−and <t>53BP1−/−</t> primary splenic B cells were stimulated with αCD40/IL4 for 96 h. (B and E) MH usage from junctions with direct and up to 10-bp MH for 5′Sμ (B) to Sγ1 and (E) to Sε DSBs are plotted as percentage of total junctions. (C, D, F, and G) Percentages of direct joins of 5′Sμ to (C) Sγ1 and (F) Sε and percentage of junctions with 4-bp or longer MH in (D) Sγ1 or (G) Sε in different genetic backgrounds are compared. Unpaired two-tailed t test was used to calculate P values for significant difference between samples (quantitative data = average ± SEM; **P ≤ 0.01, ***P ≤ 0.001). At least three independent samples were used for each experiment. SI Appendix, Table S2 lists detailed information, including junction numbers and percentage of direct versus MH-mediated joins found for each of the nonsized matched libraries analyzed to generate the data described in this figure. We also obtained highly similar results upon analyzing three replicate libraries for each genotype in which Sμ−Sγ1 or Sμ−Se junctional sequence numbers from each experimental sample were normalized via random selection to that of the experimental sample with the smallest number of recovered junctions (see SI Appendix, Fig. S1 for details).
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    1) Product Images from "DNA double-strand break response factors influence end-joining features of IgH class switch and general translocation junctions"

    Article Title: DNA double-strand break response factors influence end-joining features of IgH class switch and general translocation junctions

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1719988115

    Increased MH usage in Sμ−Sγ1 and Sμ−Sε CSR junctions from DSBR factor-deficient primary B cells. (A) Illustration of joining outcomes of Sμ region DSBs to downstream Sγ1 and Sε region breaks undergoing direct or MH-mediated joining. WT, ATM−/,−and 53BP1−/− primary splenic B cells were stimulated with αCD40/IL4 for 96 h. (B and E) MH usage from junctions with direct and up to 10-bp MH for 5′Sμ (B) to Sγ1 and (E) to Sε DSBs are plotted as percentage of total junctions. (C, D, F, and G) Percentages of direct joins of 5′Sμ to (C) Sγ1 and (F) Sε and percentage of junctions with 4-bp or longer MH in (D) Sγ1 or (G) Sε in different genetic backgrounds are compared. Unpaired two-tailed t test was used to calculate P values for significant difference between samples (quantitative data = average ± SEM; **P ≤ 0.01, ***P ≤ 0.001). At least three independent samples were used for each experiment. SI Appendix, Table S2 lists detailed information, including junction numbers and percentage of direct versus MH-mediated joins found for each of the nonsized matched libraries analyzed to generate the data described in this figure. We also obtained highly similar results upon analyzing three replicate libraries for each genotype in which Sμ−Sγ1 or Sμ−Se junctional sequence numbers from each experimental sample were normalized via random selection to that of the experimental sample with the smallest number of recovered junctions (see SI Appendix, Fig. S1 for details).
    Figure Legend Snippet: Increased MH usage in Sμ−Sγ1 and Sμ−Sε CSR junctions from DSBR factor-deficient primary B cells. (A) Illustration of joining outcomes of Sμ region DSBs to downstream Sγ1 and Sε region breaks undergoing direct or MH-mediated joining. WT, ATM−/,−and 53BP1−/− primary splenic B cells were stimulated with αCD40/IL4 for 96 h. (B and E) MH usage from junctions with direct and up to 10-bp MH for 5′Sμ (B) to Sγ1 and (E) to Sε DSBs are plotted as percentage of total junctions. (C, D, F, and G) Percentages of direct joins of 5′Sμ to (C) Sγ1 and (F) Sε and percentage of junctions with 4-bp or longer MH in (D) Sγ1 or (G) Sε in different genetic backgrounds are compared. Unpaired two-tailed t test was used to calculate P values for significant difference between samples (quantitative data = average ± SEM; **P ≤ 0.01, ***P ≤ 0.001). At least three independent samples were used for each experiment. SI Appendix, Table S2 lists detailed information, including junction numbers and percentage of direct versus MH-mediated joins found for each of the nonsized matched libraries analyzed to generate the data described in this figure. We also obtained highly similar results upon analyzing three replicate libraries for each genotype in which Sμ−Sγ1 or Sμ−Se junctional sequence numbers from each experimental sample were normalized via random selection to that of the experimental sample with the smallest number of recovered junctions (see SI Appendix, Fig. S1 for details).

    Techniques Used: Two Tailed Test, Sequencing, Selection

    Increased MH usage in Sμ−Sα CSR junctions from WT, ATM-, 53BP1-, and ligase 4-deficient CH12F3 cells. (A) Illustration of the construction of nonproductive allele-deleted CH12F3 cells and the HTGTS strategy to assay the Sμ−Sα junctions with 5′Sμ bait after 72-h stimulation with αCD40/IL4/TGF-β. (B) MH usage in junctions from joining AID-induced 5′Sμ breaks to Sα breaks in nonproductive allele-deleted WT, ATM−/−, 53BP1−/−, and ligase 4−/− CH12F3 cells were plotted as percentage of junctions with indicated length of MH over the total number of junctions in Sα region. (C and D) Percentage of (C) direct junctions and (D) junctions with 4-bp and longer MH in Sμ−Sα joining in nonproductive allele-deleted WT, ATM−/−, 53BP1−/−, and ligase 4−/− CH12F3 cells was plotted. Two-tailed t test was used for statistical analysis (quantitative data = average ± SEM; ***P ≤ 0.001). Three independent samples were used for each experiment, and details are provided in SI Appendix, Table S4.
    Figure Legend Snippet: Increased MH usage in Sμ−Sα CSR junctions from WT, ATM-, 53BP1-, and ligase 4-deficient CH12F3 cells. (A) Illustration of the construction of nonproductive allele-deleted CH12F3 cells and the HTGTS strategy to assay the Sμ−Sα junctions with 5′Sμ bait after 72-h stimulation with αCD40/IL4/TGF-β. (B) MH usage in junctions from joining AID-induced 5′Sμ breaks to Sα breaks in nonproductive allele-deleted WT, ATM−/−, 53BP1−/−, and ligase 4−/− CH12F3 cells were plotted as percentage of junctions with indicated length of MH over the total number of junctions in Sα region. (C and D) Percentage of (C) direct junctions and (D) junctions with 4-bp and longer MH in Sμ−Sα joining in nonproductive allele-deleted WT, ATM−/−, 53BP1−/−, and ligase 4−/− CH12F3 cells was plotted. Two-tailed t test was used for statistical analysis (quantitative data = average ± SEM; ***P ≤ 0.001). Three independent samples were used for each experiment, and details are provided in SI Appendix, Table S4.

    Techniques Used: Two Tailed Test

    Comparison of translocation junctions to S region or general DSBs in WT and mutant CH12F3 cells. WT, ATM-, 53BP1-, and ligase 4-deficient CH12F3 cells were stimulated with αCD40/IL4/TGF-β, and cmyc-Cas9 bait DSB was introduced 12 h after stimulation. HTGTS libraries were made with c-myc-Cas9 DSB as bait, with genomic DNA harvested 72 h poststimulation. (A) Translocation outcomes of c-myc-Cas9 DSB to genome-wide or Sμ/Sα breaks are depicted. (B and E) MH usage in junctions from c-myc-Cas9 break joining to (B) Sα breaks and (E) genome-wide breaks were plotted as percentage of junctions with indicated length of MH over the total number of junctions in the respective regions. (C, D, F, and G) Percentages of direct joins from c-myc-Cas9 break joining to (C) Sα breaks and (F) genome-wide breaks, and percentages of junctions with 4 bp and longer MH from c-myc-Cas9 break to (D) Sα breaks and (G) genome-wide breaks in different genetic backgrounds are shown. Unpaired two-tailed t test was used for statistical analysis (quantitative data = average ± SEM; n.s. means P > 0.05; ***P ≤ 0.001). Three independent samples were used for each experiment, and details are provided in SI Appendix, Table S9.
    Figure Legend Snippet: Comparison of translocation junctions to S region or general DSBs in WT and mutant CH12F3 cells. WT, ATM-, 53BP1-, and ligase 4-deficient CH12F3 cells were stimulated with αCD40/IL4/TGF-β, and cmyc-Cas9 bait DSB was introduced 12 h after stimulation. HTGTS libraries were made with c-myc-Cas9 DSB as bait, with genomic DNA harvested 72 h poststimulation. (A) Translocation outcomes of c-myc-Cas9 DSB to genome-wide or Sμ/Sα breaks are depicted. (B and E) MH usage in junctions from c-myc-Cas9 break joining to (B) Sα breaks and (E) genome-wide breaks were plotted as percentage of junctions with indicated length of MH over the total number of junctions in the respective regions. (C, D, F, and G) Percentages of direct joins from c-myc-Cas9 break joining to (C) Sα breaks and (F) genome-wide breaks, and percentages of junctions with 4 bp and longer MH from c-myc-Cas9 break to (D) Sα breaks and (G) genome-wide breaks in different genetic backgrounds are shown. Unpaired two-tailed t test was used for statistical analysis (quantitative data = average ± SEM; n.s. means P > 0.05; ***P ≤ 0.001). Three independent samples were used for each experiment, and details are provided in SI Appendix, Table S9.

    Techniques Used: Translocation Assay, Mutagenesis, Genome Wide, Two Tailed Test

    rabbit polyclonal anti 53bp1 novus  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc rabbit polyclonal anti 53bp1 novus
    The NTD but not the CTD of MLH1 rescues TSI caused by MLH1 deficiency. ( A ) Schematic representation of functional domains in MLH1 and its deletion mutants. Purple box: ATPase domain. Blue box: MutSα-interacting domain. Green box: EXO1/PMS2/MLH3-binding domain. ( B ) Truncated FLAG-MLH1 proteins localize in nucleus in untreated cells. WT and truncated FLAG-MLH1 were stably expressed with retroviral transduction. FLAG antibody (green) was used for IF to detect FLAG-tagged WT MLH1 and mutants. ( C ) Recruitment of full-length MLH1 (WT) and NTD but not CTD of MLH1 to DNA damage sites induced by etoposide treatment (0.3 μM, 2 h). After etoposide treatment, IF was performed with <t>anti-53BP1</t> (red) and anti-FLAG (green) in cells expressing WT or mutated FLAG-MLH1. Representative co-localizations of <t>53BP1</t> and FLAG-MLH1 were labeled with numbers and then enlarged in insets. ( D ) Percentage of cells with MLH1/53BP1 co-localization (>5 colocalizations per cell). ( E ) Frequency of TSI measured in HeLa shLUC and shMLH1 cells with concurrent expression of vector (v), WT-MLH1, NTD, or CTD. Cells were treated with 3 μM etoposide for 2 h and then recovered for 24 h prior to FISH. In each experiment, >1500 chromosomes from each sample were analyzed and each experiment was repeated with three independent replicates.
    Rabbit Polyclonal Anti 53bp1 Novus, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Human MLH1 suppresses the insertion of telomeric sequences at intra-chromosomal sites in telomerase-expressing cells"

    Article Title: Human MLH1 suppresses the insertion of telomeric sequences at intra-chromosomal sites in telomerase-expressing cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1170

    The NTD but not the CTD of MLH1 rescues TSI caused by MLH1 deficiency. ( A ) Schematic representation of functional domains in MLH1 and its deletion mutants. Purple box: ATPase domain. Blue box: MutSα-interacting domain. Green box: EXO1/PMS2/MLH3-binding domain. ( B ) Truncated FLAG-MLH1 proteins localize in nucleus in untreated cells. WT and truncated FLAG-MLH1 were stably expressed with retroviral transduction. FLAG antibody (green) was used for IF to detect FLAG-tagged WT MLH1 and mutants. ( C ) Recruitment of full-length MLH1 (WT) and NTD but not CTD of MLH1 to DNA damage sites induced by etoposide treatment (0.3 μM, 2 h). After etoposide treatment, IF was performed with anti-53BP1 (red) and anti-FLAG (green) in cells expressing WT or mutated FLAG-MLH1. Representative co-localizations of 53BP1 and FLAG-MLH1 were labeled with numbers and then enlarged in insets. ( D ) Percentage of cells with MLH1/53BP1 co-localization (>5 colocalizations per cell). ( E ) Frequency of TSI measured in HeLa shLUC and shMLH1 cells with concurrent expression of vector (v), WT-MLH1, NTD, or CTD. Cells were treated with 3 μM etoposide for 2 h and then recovered for 24 h prior to FISH. In each experiment, >1500 chromosomes from each sample were analyzed and each experiment was repeated with three independent replicates.
    Figure Legend Snippet: The NTD but not the CTD of MLH1 rescues TSI caused by MLH1 deficiency. ( A ) Schematic representation of functional domains in MLH1 and its deletion mutants. Purple box: ATPase domain. Blue box: MutSα-interacting domain. Green box: EXO1/PMS2/MLH3-binding domain. ( B ) Truncated FLAG-MLH1 proteins localize in nucleus in untreated cells. WT and truncated FLAG-MLH1 were stably expressed with retroviral transduction. FLAG antibody (green) was used for IF to detect FLAG-tagged WT MLH1 and mutants. ( C ) Recruitment of full-length MLH1 (WT) and NTD but not CTD of MLH1 to DNA damage sites induced by etoposide treatment (0.3 μM, 2 h). After etoposide treatment, IF was performed with anti-53BP1 (red) and anti-FLAG (green) in cells expressing WT or mutated FLAG-MLH1. Representative co-localizations of 53BP1 and FLAG-MLH1 were labeled with numbers and then enlarged in insets. ( D ) Percentage of cells with MLH1/53BP1 co-localization (>5 colocalizations per cell). ( E ) Frequency of TSI measured in HeLa shLUC and shMLH1 cells with concurrent expression of vector (v), WT-MLH1, NTD, or CTD. Cells were treated with 3 μM etoposide for 2 h and then recovered for 24 h prior to FISH. In each experiment, >1500 chromosomes from each sample were analyzed and each experiment was repeated with three independent replicates.

    Techniques Used: Functional Assay, Binding Assay, Stable Transfection, Transduction, Expressing, Labeling, Plasmid Preparation

    53bp1 rabbit polyclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 53bp1 rabbit polyclonal antibody
    53bp1 Rabbit Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc 53bp1
    Recruitment of various DDR proteins to lacO arrays in U2OS 40–2-6 ER-LacI cells. (A) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT for 2 h were double immunostained with the indicated antibodies and counterstained with DAPI. ssDNA was stained with anti-BrdU antibody under nondenaturing conditions. For detection of total ssDNA, DNA was labeled for 24 h with 10 µM BrdU. For detection of nascent-strand ssDNA, newly synthesized DNA was labeled with BrdU during treatment with 4-OHT. For detection of parental-strand ssDNA, DNA was labeled with 10 µM BrdU for 20 h, followed by a chase in fresh medium without BrdU for 2 h before addition of 4-OHT. Representative images are shown, and the colocalization frequencies are shown in . Yellow arrows indicate colocalization of DDR proteins with the foci of HA-LacI, and white arrows indicate noncolocalization. Scale bars, 10 µm. (B) The antibody used in this study can detect bleomycin-induced RAD51 foci. U2OS 40–2-6 ER-LacI cells were treated with 8 µM Bleomycin for 18 h, then immunostained with anti-RAD51 antibody, followed by DAPI staining. Representative images are shown. Scale bar, 10 µm. (C) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT or a control vehicle (EtOH) for 2 h were subjected to ChIP-quantitative PCR analysis using the indicated antibodies. Enrichment of the lacO sequences is shown as the percent of input DNA. Means ± SD are shown ( n = 6 for control rabbit IgG; n = 7 for LacI and <t>53BP1).</t> *, P < 0.05; ***, P < 0.001; n.s., not significant (two-tailed Student’s t test).
    53bp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc α 53bp1 antibody
    Increased MH usage in Sμ−Sγ1 and Sμ−Sε CSR junctions from DSBR factor-deficient primary B cells. (A) Illustration of joining outcomes of Sμ region DSBs to downstream Sγ1 and Sε region breaks undergoing direct or MH-mediated joining. WT, ATM−/,−and <t>53BP1−/−</t> primary splenic B cells were stimulated with αCD40/IL4 for 96 h. (B and E) MH usage from junctions with direct and up to 10-bp MH for 5′Sμ (B) to Sγ1 and (E) to Sε DSBs are plotted as percentage of total junctions. (C, D, F, and G) Percentages of direct joins of 5′Sμ to (C) Sγ1 and (F) Sε and percentage of junctions with 4-bp or longer MH in (D) Sγ1 or (G) Sε in different genetic backgrounds are compared. Unpaired two-tailed t test was used to calculate P values for significant difference between samples (quantitative data = average ± SEM; **P ≤ 0.01, ***P ≤ 0.001). At least three independent samples were used for each experiment. SI Appendix, Table S2 lists detailed information, including junction numbers and percentage of direct versus MH-mediated joins found for each of the nonsized matched libraries analyzed to generate the data described in this figure. We also obtained highly similar results upon analyzing three replicate libraries for each genotype in which Sμ−Sγ1 or Sμ−Se junctional sequence numbers from each experimental sample were normalized via random selection to that of the experimental sample with the smallest number of recovered junctions (see SI Appendix, Fig. S1 for details).
    α 53bp1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit polyclonal anti 53bp1 novus
    The NTD but not the CTD of MLH1 rescues TSI caused by MLH1 deficiency. ( A ) Schematic representation of functional domains in MLH1 and its deletion mutants. Purple box: ATPase domain. Blue box: MutSα-interacting domain. Green box: EXO1/PMS2/MLH3-binding domain. ( B ) Truncated FLAG-MLH1 proteins localize in nucleus in untreated cells. WT and truncated FLAG-MLH1 were stably expressed with retroviral transduction. FLAG antibody (green) was used for IF to detect FLAG-tagged WT MLH1 and mutants. ( C ) Recruitment of full-length MLH1 (WT) and NTD but not CTD of MLH1 to DNA damage sites induced by etoposide treatment (0.3 μM, 2 h). After etoposide treatment, IF was performed with <t>anti-53BP1</t> (red) and anti-FLAG (green) in cells expressing WT or mutated FLAG-MLH1. Representative co-localizations of <t>53BP1</t> and FLAG-MLH1 were labeled with numbers and then enlarged in insets. ( D ) Percentage of cells with MLH1/53BP1 co-localization (>5 colocalizations per cell). ( E ) Frequency of TSI measured in HeLa shLUC and shMLH1 cells with concurrent expression of vector (v), WT-MLH1, NTD, or CTD. Cells were treated with 3 μM etoposide for 2 h and then recovered for 24 h prior to FISH. In each experiment, >1500 chromosomes from each sample were analyzed and each experiment was repeated with three independent replicates.
    Rabbit Polyclonal Anti 53bp1 Novus, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc 53bp1 rabbit polyclonal antibody
    The NTD but not the CTD of MLH1 rescues TSI caused by MLH1 deficiency. ( A ) Schematic representation of functional domains in MLH1 and its deletion mutants. Purple box: ATPase domain. Blue box: MutSα-interacting domain. Green box: EXO1/PMS2/MLH3-binding domain. ( B ) Truncated FLAG-MLH1 proteins localize in nucleus in untreated cells. WT and truncated FLAG-MLH1 were stably expressed with retroviral transduction. FLAG antibody (green) was used for IF to detect FLAG-tagged WT MLH1 and mutants. ( C ) Recruitment of full-length MLH1 (WT) and NTD but not CTD of MLH1 to DNA damage sites induced by etoposide treatment (0.3 μM, 2 h). After etoposide treatment, IF was performed with <t>anti-53BP1</t> (red) and anti-FLAG (green) in cells expressing WT or mutated FLAG-MLH1. Representative co-localizations of <t>53BP1</t> and FLAG-MLH1 were labeled with numbers and then enlarged in insets. ( D ) Percentage of cells with MLH1/53BP1 co-localization (>5 colocalizations per cell). ( E ) Frequency of TSI measured in HeLa shLUC and shMLH1 cells with concurrent expression of vector (v), WT-MLH1, NTD, or CTD. Cells were treated with 3 μM etoposide for 2 h and then recovered for 24 h prior to FISH. In each experiment, >1500 chromosomes from each sample were analyzed and each experiment was repeated with three independent replicates.
    53bp1 Rabbit Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    Image Search Results


    Recruitment of various DDR proteins to lacO arrays in U2OS 40–2-6 ER-LacI cells. (A) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT for 2 h were double immunostained with the indicated antibodies and counterstained with DAPI. ssDNA was stained with anti-BrdU antibody under nondenaturing conditions. For detection of total ssDNA, DNA was labeled for 24 h with 10 µM BrdU. For detection of nascent-strand ssDNA, newly synthesized DNA was labeled with BrdU during treatment with 4-OHT. For detection of parental-strand ssDNA, DNA was labeled with 10 µM BrdU for 20 h, followed by a chase in fresh medium without BrdU for 2 h before addition of 4-OHT. Representative images are shown, and the colocalization frequencies are shown in . Yellow arrows indicate colocalization of DDR proteins with the foci of HA-LacI, and white arrows indicate noncolocalization. Scale bars, 10 µm. (B) The antibody used in this study can detect bleomycin-induced RAD51 foci. U2OS 40–2-6 ER-LacI cells were treated with 8 µM Bleomycin for 18 h, then immunostained with anti-RAD51 antibody, followed by DAPI staining. Representative images are shown. Scale bar, 10 µm. (C) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT or a control vehicle (EtOH) for 2 h were subjected to ChIP-quantitative PCR analysis using the indicated antibodies. Enrichment of the lacO sequences is shown as the percent of input DNA. Means ± SD are shown ( n = 6 for control rabbit IgG; n = 7 for LacI and 53BP1). *, P < 0.05; ***, P < 0.001; n.s., not significant (two-tailed Student’s t test).

    Journal: The Journal of Cell Biology

    Article Title: SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions

    doi: 10.1083/jcb.202003148

    Figure Lengend Snippet: Recruitment of various DDR proteins to lacO arrays in U2OS 40–2-6 ER-LacI cells. (A) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT for 2 h were double immunostained with the indicated antibodies and counterstained with DAPI. ssDNA was stained with anti-BrdU antibody under nondenaturing conditions. For detection of total ssDNA, DNA was labeled for 24 h with 10 µM BrdU. For detection of nascent-strand ssDNA, newly synthesized DNA was labeled with BrdU during treatment with 4-OHT. For detection of parental-strand ssDNA, DNA was labeled with 10 µM BrdU for 20 h, followed by a chase in fresh medium without BrdU for 2 h before addition of 4-OHT. Representative images are shown, and the colocalization frequencies are shown in . Yellow arrows indicate colocalization of DDR proteins with the foci of HA-LacI, and white arrows indicate noncolocalization. Scale bars, 10 µm. (B) The antibody used in this study can detect bleomycin-induced RAD51 foci. U2OS 40–2-6 ER-LacI cells were treated with 8 µM Bleomycin for 18 h, then immunostained with anti-RAD51 antibody, followed by DAPI staining. Representative images are shown. Scale bar, 10 µm. (C) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT or a control vehicle (EtOH) for 2 h were subjected to ChIP-quantitative PCR analysis using the indicated antibodies. Enrichment of the lacO sequences is shown as the percent of input DNA. Means ± SD are shown ( n = 6 for control rabbit IgG; n = 7 for LacI and 53BP1). *, P < 0.05; ***, P < 0.001; n.s., not significant (two-tailed Student’s t test).

    Article Snippet: Other antibodies used in this study were as follows: ATR (Santa Cruz; Goat, sc1887), ATRIP (Cell Signaling; Rabbit, #2737), RPA34 (Calbiochem; Mouse, NA19L), TOPBP1 (Abcam; Rabbit, ab2402), FANCD2 (Novus; Rabbit, NB100-182), LacI (clone 9A5, Merck, Mouse, #05-503), HA (clone 3F10; Roche; Rat, 11867423001), Chk1 (Santa Cruz; Mouse, sc-8408), phosphorylated Chk1 (Ser345; Cell Signaling; Rabbit, #2348S), phosphorylated ATM (Ser1981; Rockland; Mouse, 200–301-400; Abcam; Rabbit, ab81292), 53BP1 (Novus; Rabbit, NB100-904), RAD51 (BioAcademia; Rabbit, 70–012), RAD52 (BioAcademia; Rabbit, 70–015), BrdU (clone 3D4 and B44; BD PharMingen; Mouse, 555627 and 347580, respectively), Cyclin A (Santa Cruz; Rabbit, sc-596), Cyclin E (Santa Cruz; Mouse, sc-247), SLX4 (Novus; Rabbit, NBP1-28680), XPF (Bethyl; Rabbit, A301-315A), MUS81 (Abcam; Mouse, ab14387), SLX1 (Atlas Antibodies; Rabbit, HPA047038), PICH (clone 142–26-3, Merck, Mouse, 04–1540), goat normal IgG (Chemicon; PP40), mouse normal IgG (Southern Biotech; 0107–01), and rabbit normal IgG (Dako; X0903).

    Techniques: Staining, Labeling, Synthesized, Real-time Polymerase Chain Reaction, Two Tailed Test

    A model of the fate of underreplicated DNA regions in the lacO array. Even in the absence of LacI binding, the lacO array is a difficult-to-replicate region, like other repeat sequences, and contains underreplicated lesions (∼60% on the basis of the data shown in ), which persist until late G2/M phase. Replication stress by LacI binding to the array further increases the frequency of cells with underreplicated DNA lesions (∼90%). To try to complete replication of the array during S phase, cells activate the DDR, in which one-ended DSB is first generated by SLX4–XPF–mediated DNA cleavage. Excess ssDNA is then generated by unidentified exonucleases, which in turn recruit ATR, FANCD2, and RAD52. ATR and FANCD2 are interdependently recruited. The DDR may promote completion of replication, because SLX4 and ATR inhibition exacerbates mitotic abnormality induced by LacI. The underreplicated intermediates persisting until late G2/M phase are processed by MUS81-mediated cleavage in early mitosis. Note that cleaved strands are arbitrary in the figure, because it remains unclear whether the cleavage occurs in the leading or lagging strand templates. Cleavage at lacO can promote MiDAS (∼20% in the absence or presence of LacI; ) or end joining that results in deletion of the loci (∼30% in the absence of LacI and ∼50% in the presence of LacI; ). A fraction of the lacO arrays escape from the cleavage and remain underreplicated until anaphase, leading to the anaphase abnormal lacO structure (∼5% in the absence of LacI and ∼20% in the presence of LacI; ) and the formation of 53BP1 NBs in daughter G1 cells (∼5% in the absence of LacI and ∼20% in the presence of LacI; ).

    Journal: The Journal of Cell Biology

    Article Title: SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions

    doi: 10.1083/jcb.202003148

    Figure Lengend Snippet: A model of the fate of underreplicated DNA regions in the lacO array. Even in the absence of LacI binding, the lacO array is a difficult-to-replicate region, like other repeat sequences, and contains underreplicated lesions (∼60% on the basis of the data shown in ), which persist until late G2/M phase. Replication stress by LacI binding to the array further increases the frequency of cells with underreplicated DNA lesions (∼90%). To try to complete replication of the array during S phase, cells activate the DDR, in which one-ended DSB is first generated by SLX4–XPF–mediated DNA cleavage. Excess ssDNA is then generated by unidentified exonucleases, which in turn recruit ATR, FANCD2, and RAD52. ATR and FANCD2 are interdependently recruited. The DDR may promote completion of replication, because SLX4 and ATR inhibition exacerbates mitotic abnormality induced by LacI. The underreplicated intermediates persisting until late G2/M phase are processed by MUS81-mediated cleavage in early mitosis. Note that cleaved strands are arbitrary in the figure, because it remains unclear whether the cleavage occurs in the leading or lagging strand templates. Cleavage at lacO can promote MiDAS (∼20% in the absence or presence of LacI; ) or end joining that results in deletion of the loci (∼30% in the absence of LacI and ∼50% in the presence of LacI; ). A fraction of the lacO arrays escape from the cleavage and remain underreplicated until anaphase, leading to the anaphase abnormal lacO structure (∼5% in the absence of LacI and ∼20% in the presence of LacI; ) and the formation of 53BP1 NBs in daughter G1 cells (∼5% in the absence of LacI and ∼20% in the presence of LacI; ).

    Article Snippet: Other antibodies used in this study were as follows: ATR (Santa Cruz; Goat, sc1887), ATRIP (Cell Signaling; Rabbit, #2737), RPA34 (Calbiochem; Mouse, NA19L), TOPBP1 (Abcam; Rabbit, ab2402), FANCD2 (Novus; Rabbit, NB100-182), LacI (clone 9A5, Merck, Mouse, #05-503), HA (clone 3F10; Roche; Rat, 11867423001), Chk1 (Santa Cruz; Mouse, sc-8408), phosphorylated Chk1 (Ser345; Cell Signaling; Rabbit, #2348S), phosphorylated ATM (Ser1981; Rockland; Mouse, 200–301-400; Abcam; Rabbit, ab81292), 53BP1 (Novus; Rabbit, NB100-904), RAD51 (BioAcademia; Rabbit, 70–012), RAD52 (BioAcademia; Rabbit, 70–015), BrdU (clone 3D4 and B44; BD PharMingen; Mouse, 555627 and 347580, respectively), Cyclin A (Santa Cruz; Rabbit, sc-596), Cyclin E (Santa Cruz; Mouse, sc-247), SLX4 (Novus; Rabbit, NBP1-28680), XPF (Bethyl; Rabbit, A301-315A), MUS81 (Abcam; Mouse, ab14387), SLX1 (Atlas Antibodies; Rabbit, HPA047038), PICH (clone 142–26-3, Merck, Mouse, 04–1540), goat normal IgG (Chemicon; PP40), mouse normal IgG (Southern Biotech; 0107–01), and rabbit normal IgG (Dako; X0903).

    Techniques: Binding Assay, Generated, Inhibition

    LacI binding in S phase promotes a UFB-like anaphase abnormal lacO array and underreplicated DNA lesions in daughter cells. (A and B) U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase by treatment with 7 µM RO-3306 for 20 h and 1 µM 4-OHT (for LacI induction) for 20 h or the last 4 h. The G2-arrested cells were then released into fresh medium for 45 min to enable progression into anaphase and immunostained with the indicated antibodies, followed by DAPI staining and analysis. (A) Representative images of anaphase cells. Yellow arrows indicate DAPI-negative anaphase abnormal lacO arrays colocalized with RPA and PICH. White arrows denote normally segregated lacO arrays. Scale bars, 10 µm. (B) The graph indicates frequencies of anaphase abnormal lacO per anaphase cell. Values are sum scores from two independent experiments. ***, P < 0.001 (χ 2 test). Individual data points from the two independent experiments are also shown. (C–E) U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase and released into G1 phase as described in . Cells were double immunostained with anti-LacI and anti-53BP1 antibodies, followed by DAPI staining and analysis. (C) Representative images of twin 53BP1 NBs (indicated by arrows) that colocalize with the symmetrical LacI foci in the sister cells. Scale bar, 10 µm. (D) A diagram depicting different patterns of 53BP1 NBs in the sister cells with symmetrical LacI foci. (E) The graph indicates frequencies of sister pairs with twin 53BP1 NBs as a percentage of sister pairs harboring symmetrical LacI foci. Values are sum scores from two independent experiments. **, P < 0.01 (χ 2 test). Individual data points from the two independent experiments are also shown.

    Journal: The Journal of Cell Biology

    Article Title: SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions

    doi: 10.1083/jcb.202003148

    Figure Lengend Snippet: LacI binding in S phase promotes a UFB-like anaphase abnormal lacO array and underreplicated DNA lesions in daughter cells. (A and B) U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase by treatment with 7 µM RO-3306 for 20 h and 1 µM 4-OHT (for LacI induction) for 20 h or the last 4 h. The G2-arrested cells were then released into fresh medium for 45 min to enable progression into anaphase and immunostained with the indicated antibodies, followed by DAPI staining and analysis. (A) Representative images of anaphase cells. Yellow arrows indicate DAPI-negative anaphase abnormal lacO arrays colocalized with RPA and PICH. White arrows denote normally segregated lacO arrays. Scale bars, 10 µm. (B) The graph indicates frequencies of anaphase abnormal lacO per anaphase cell. Values are sum scores from two independent experiments. ***, P < 0.001 (χ 2 test). Individual data points from the two independent experiments are also shown. (C–E) U2OS 40–2-6 ER-LacI cells were synchronized in late G2 phase and released into G1 phase as described in . Cells were double immunostained with anti-LacI and anti-53BP1 antibodies, followed by DAPI staining and analysis. (C) Representative images of twin 53BP1 NBs (indicated by arrows) that colocalize with the symmetrical LacI foci in the sister cells. Scale bar, 10 µm. (D) A diagram depicting different patterns of 53BP1 NBs in the sister cells with symmetrical LacI foci. (E) The graph indicates frequencies of sister pairs with twin 53BP1 NBs as a percentage of sister pairs harboring symmetrical LacI foci. Values are sum scores from two independent experiments. **, P < 0.01 (χ 2 test). Individual data points from the two independent experiments are also shown.

    Article Snippet: Other antibodies used in this study were as follows: ATR (Santa Cruz; Goat, sc1887), ATRIP (Cell Signaling; Rabbit, #2737), RPA34 (Calbiochem; Mouse, NA19L), TOPBP1 (Abcam; Rabbit, ab2402), FANCD2 (Novus; Rabbit, NB100-182), LacI (clone 9A5, Merck, Mouse, #05-503), HA (clone 3F10; Roche; Rat, 11867423001), Chk1 (Santa Cruz; Mouse, sc-8408), phosphorylated Chk1 (Ser345; Cell Signaling; Rabbit, #2348S), phosphorylated ATM (Ser1981; Rockland; Mouse, 200–301-400; Abcam; Rabbit, ab81292), 53BP1 (Novus; Rabbit, NB100-904), RAD51 (BioAcademia; Rabbit, 70–012), RAD52 (BioAcademia; Rabbit, 70–015), BrdU (clone 3D4 and B44; BD PharMingen; Mouse, 555627 and 347580, respectively), Cyclin A (Santa Cruz; Rabbit, sc-596), Cyclin E (Santa Cruz; Mouse, sc-247), SLX4 (Novus; Rabbit, NBP1-28680), XPF (Bethyl; Rabbit, A301-315A), MUS81 (Abcam; Mouse, ab14387), SLX1 (Atlas Antibodies; Rabbit, HPA047038), PICH (clone 142–26-3, Merck, Mouse, 04–1540), goat normal IgG (Chemicon; PP40), mouse normal IgG (Southern Biotech; 0107–01), and rabbit normal IgG (Dako; X0903).

    Techniques: Binding Assay, Staining

    Increased MH usage in Sμ−Sγ1 and Sμ−Sε CSR junctions from DSBR factor-deficient primary B cells. (A) Illustration of joining outcomes of Sμ region DSBs to downstream Sγ1 and Sε region breaks undergoing direct or MH-mediated joining. WT, ATM−/,−and 53BP1−/− primary splenic B cells were stimulated with αCD40/IL4 for 96 h. (B and E) MH usage from junctions with direct and up to 10-bp MH for 5′Sμ (B) to Sγ1 and (E) to Sε DSBs are plotted as percentage of total junctions. (C, D, F, and G) Percentages of direct joins of 5′Sμ to (C) Sγ1 and (F) Sε and percentage of junctions with 4-bp or longer MH in (D) Sγ1 or (G) Sε in different genetic backgrounds are compared. Unpaired two-tailed t test was used to calculate P values for significant difference between samples (quantitative data = average ± SEM; **P ≤ 0.01, ***P ≤ 0.001). At least three independent samples were used for each experiment. SI Appendix, Table S2 lists detailed information, including junction numbers and percentage of direct versus MH-mediated joins found for each of the nonsized matched libraries analyzed to generate the data described in this figure. We also obtained highly similar results upon analyzing three replicate libraries for each genotype in which Sμ−Sγ1 or Sμ−Se junctional sequence numbers from each experimental sample were normalized via random selection to that of the experimental sample with the smallest number of recovered junctions (see SI Appendix, Fig. S1 for details).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: DNA double-strand break response factors influence end-joining features of IgH class switch and general translocation junctions

    doi: 10.1073/pnas.1719988115

    Figure Lengend Snippet: Increased MH usage in Sμ−Sγ1 and Sμ−Sε CSR junctions from DSBR factor-deficient primary B cells. (A) Illustration of joining outcomes of Sμ region DSBs to downstream Sγ1 and Sε region breaks undergoing direct or MH-mediated joining. WT, ATM−/,−and 53BP1−/− primary splenic B cells were stimulated with αCD40/IL4 for 96 h. (B and E) MH usage from junctions with direct and up to 10-bp MH for 5′Sμ (B) to Sγ1 and (E) to Sε DSBs are plotted as percentage of total junctions. (C, D, F, and G) Percentages of direct joins of 5′Sμ to (C) Sγ1 and (F) Sε and percentage of junctions with 4-bp or longer MH in (D) Sγ1 or (G) Sε in different genetic backgrounds are compared. Unpaired two-tailed t test was used to calculate P values for significant difference between samples (quantitative data = average ± SEM; **P ≤ 0.01, ***P ≤ 0.001). At least three independent samples were used for each experiment. SI Appendix, Table S2 lists detailed information, including junction numbers and percentage of direct versus MH-mediated joins found for each of the nonsized matched libraries analyzed to generate the data described in this figure. We also obtained highly similar results upon analyzing three replicate libraries for each genotype in which Sμ−Sγ1 or Sμ−Se junctional sequence numbers from each experimental sample were normalized via random selection to that of the experimental sample with the smallest number of recovered junctions (see SI Appendix, Fig. S1 for details).

    Article Snippet: The 53BP1 −/− CH12F3 cells were obtained similarly as above, and two independent clones generated were confirmed by Southern blot and Western blot with α-53BP1 antibody (#4908; Cell Signaling Technology).

    Techniques: Two Tailed Test, Sequencing, Selection

    Increased MH usage in Sμ−Sα CSR junctions from WT, ATM-, 53BP1-, and ligase 4-deficient CH12F3 cells. (A) Illustration of the construction of nonproductive allele-deleted CH12F3 cells and the HTGTS strategy to assay the Sμ−Sα junctions with 5′Sμ bait after 72-h stimulation with αCD40/IL4/TGF-β. (B) MH usage in junctions from joining AID-induced 5′Sμ breaks to Sα breaks in nonproductive allele-deleted WT, ATM−/−, 53BP1−/−, and ligase 4−/− CH12F3 cells were plotted as percentage of junctions with indicated length of MH over the total number of junctions in Sα region. (C and D) Percentage of (C) direct junctions and (D) junctions with 4-bp and longer MH in Sμ−Sα joining in nonproductive allele-deleted WT, ATM−/−, 53BP1−/−, and ligase 4−/− CH12F3 cells was plotted. Two-tailed t test was used for statistical analysis (quantitative data = average ± SEM; ***P ≤ 0.001). Three independent samples were used for each experiment, and details are provided in SI Appendix, Table S4.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: DNA double-strand break response factors influence end-joining features of IgH class switch and general translocation junctions

    doi: 10.1073/pnas.1719988115

    Figure Lengend Snippet: Increased MH usage in Sμ−Sα CSR junctions from WT, ATM-, 53BP1-, and ligase 4-deficient CH12F3 cells. (A) Illustration of the construction of nonproductive allele-deleted CH12F3 cells and the HTGTS strategy to assay the Sμ−Sα junctions with 5′Sμ bait after 72-h stimulation with αCD40/IL4/TGF-β. (B) MH usage in junctions from joining AID-induced 5′Sμ breaks to Sα breaks in nonproductive allele-deleted WT, ATM−/−, 53BP1−/−, and ligase 4−/− CH12F3 cells were plotted as percentage of junctions with indicated length of MH over the total number of junctions in Sα region. (C and D) Percentage of (C) direct junctions and (D) junctions with 4-bp and longer MH in Sμ−Sα joining in nonproductive allele-deleted WT, ATM−/−, 53BP1−/−, and ligase 4−/− CH12F3 cells was plotted. Two-tailed t test was used for statistical analysis (quantitative data = average ± SEM; ***P ≤ 0.001). Three independent samples were used for each experiment, and details are provided in SI Appendix, Table S4.

    Article Snippet: The 53BP1 −/− CH12F3 cells were obtained similarly as above, and two independent clones generated were confirmed by Southern blot and Western blot with α-53BP1 antibody (#4908; Cell Signaling Technology).

    Techniques: Two Tailed Test

    Comparison of translocation junctions to S region or general DSBs in WT and mutant CH12F3 cells. WT, ATM-, 53BP1-, and ligase 4-deficient CH12F3 cells were stimulated with αCD40/IL4/TGF-β, and cmyc-Cas9 bait DSB was introduced 12 h after stimulation. HTGTS libraries were made with c-myc-Cas9 DSB as bait, with genomic DNA harvested 72 h poststimulation. (A) Translocation outcomes of c-myc-Cas9 DSB to genome-wide or Sμ/Sα breaks are depicted. (B and E) MH usage in junctions from c-myc-Cas9 break joining to (B) Sα breaks and (E) genome-wide breaks were plotted as percentage of junctions with indicated length of MH over the total number of junctions in the respective regions. (C, D, F, and G) Percentages of direct joins from c-myc-Cas9 break joining to (C) Sα breaks and (F) genome-wide breaks, and percentages of junctions with 4 bp and longer MH from c-myc-Cas9 break to (D) Sα breaks and (G) genome-wide breaks in different genetic backgrounds are shown. Unpaired two-tailed t test was used for statistical analysis (quantitative data = average ± SEM; n.s. means P > 0.05; ***P ≤ 0.001). Three independent samples were used for each experiment, and details are provided in SI Appendix, Table S9.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: DNA double-strand break response factors influence end-joining features of IgH class switch and general translocation junctions

    doi: 10.1073/pnas.1719988115

    Figure Lengend Snippet: Comparison of translocation junctions to S region or general DSBs in WT and mutant CH12F3 cells. WT, ATM-, 53BP1-, and ligase 4-deficient CH12F3 cells were stimulated with αCD40/IL4/TGF-β, and cmyc-Cas9 bait DSB was introduced 12 h after stimulation. HTGTS libraries were made with c-myc-Cas9 DSB as bait, with genomic DNA harvested 72 h poststimulation. (A) Translocation outcomes of c-myc-Cas9 DSB to genome-wide or Sμ/Sα breaks are depicted. (B and E) MH usage in junctions from c-myc-Cas9 break joining to (B) Sα breaks and (E) genome-wide breaks were plotted as percentage of junctions with indicated length of MH over the total number of junctions in the respective regions. (C, D, F, and G) Percentages of direct joins from c-myc-Cas9 break joining to (C) Sα breaks and (F) genome-wide breaks, and percentages of junctions with 4 bp and longer MH from c-myc-Cas9 break to (D) Sα breaks and (G) genome-wide breaks in different genetic backgrounds are shown. Unpaired two-tailed t test was used for statistical analysis (quantitative data = average ± SEM; n.s. means P > 0.05; ***P ≤ 0.001). Three independent samples were used for each experiment, and details are provided in SI Appendix, Table S9.

    Article Snippet: The 53BP1 −/− CH12F3 cells were obtained similarly as above, and two independent clones generated were confirmed by Southern blot and Western blot with α-53BP1 antibody (#4908; Cell Signaling Technology).

    Techniques: Translocation Assay, Mutagenesis, Genome Wide, Two Tailed Test

    The NTD but not the CTD of MLH1 rescues TSI caused by MLH1 deficiency. ( A ) Schematic representation of functional domains in MLH1 and its deletion mutants. Purple box: ATPase domain. Blue box: MutSα-interacting domain. Green box: EXO1/PMS2/MLH3-binding domain. ( B ) Truncated FLAG-MLH1 proteins localize in nucleus in untreated cells. WT and truncated FLAG-MLH1 were stably expressed with retroviral transduction. FLAG antibody (green) was used for IF to detect FLAG-tagged WT MLH1 and mutants. ( C ) Recruitment of full-length MLH1 (WT) and NTD but not CTD of MLH1 to DNA damage sites induced by etoposide treatment (0.3 μM, 2 h). After etoposide treatment, IF was performed with anti-53BP1 (red) and anti-FLAG (green) in cells expressing WT or mutated FLAG-MLH1. Representative co-localizations of 53BP1 and FLAG-MLH1 were labeled with numbers and then enlarged in insets. ( D ) Percentage of cells with MLH1/53BP1 co-localization (>5 colocalizations per cell). ( E ) Frequency of TSI measured in HeLa shLUC and shMLH1 cells with concurrent expression of vector (v), WT-MLH1, NTD, or CTD. Cells were treated with 3 μM etoposide for 2 h and then recovered for 24 h prior to FISH. In each experiment, >1500 chromosomes from each sample were analyzed and each experiment was repeated with three independent replicates.

    Journal: Nucleic Acids Research

    Article Title: Human MLH1 suppresses the insertion of telomeric sequences at intra-chromosomal sites in telomerase-expressing cells

    doi: 10.1093/nar/gkw1170

    Figure Lengend Snippet: The NTD but not the CTD of MLH1 rescues TSI caused by MLH1 deficiency. ( A ) Schematic representation of functional domains in MLH1 and its deletion mutants. Purple box: ATPase domain. Blue box: MutSα-interacting domain. Green box: EXO1/PMS2/MLH3-binding domain. ( B ) Truncated FLAG-MLH1 proteins localize in nucleus in untreated cells. WT and truncated FLAG-MLH1 were stably expressed with retroviral transduction. FLAG antibody (green) was used for IF to detect FLAG-tagged WT MLH1 and mutants. ( C ) Recruitment of full-length MLH1 (WT) and NTD but not CTD of MLH1 to DNA damage sites induced by etoposide treatment (0.3 μM, 2 h). After etoposide treatment, IF was performed with anti-53BP1 (red) and anti-FLAG (green) in cells expressing WT or mutated FLAG-MLH1. Representative co-localizations of 53BP1 and FLAG-MLH1 were labeled with numbers and then enlarged in insets. ( D ) Percentage of cells with MLH1/53BP1 co-localization (>5 colocalizations per cell). ( E ) Frequency of TSI measured in HeLa shLUC and shMLH1 cells with concurrent expression of vector (v), WT-MLH1, NTD, or CTD. Cells were treated with 3 μM etoposide for 2 h and then recovered for 24 h prior to FISH. In each experiment, >1500 chromosomes from each sample were analyzed and each experiment was repeated with three independent replicates.

    Article Snippet: The following primary antibodies were used: monoclonal anti-MLH1 (ThermoFisher), rabbit polyclonal anti-FLAG (Cell Signaling), monoclonal mouse anti-actin (BD Biosciences), rabbit polyclonal anti-53BP1 (Novus), rabbit anti-phospho-Chk1 (Ser317) (Cell Signaling), rabbit anti-phospho-Chk2 (Thr68) (Cell Signaling), rabbit anti-Chk1(Cell Signaling), rabbit anti-Chk2 (Cell Signaling), mouse anti-retinoblastoma (Rb) (BD Pharmingen), mouse anti-p53 (Santa Cruz).

    Techniques: Functional Assay, Binding Assay, Stable Transfection, Transduction, Expressing, Labeling, Plasmid Preparation