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u2os cells expressing ostir1 9myc  (Addgene inc)


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    Addgene inc u2os cells expressing ostir1 9myc
    Fig. 4 ATAD5 promotes generation of single-stranded DNA-associated breaks in response to replication stress. a <t>U2OS</t> cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 or 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. b, c U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 h before fixation. The fixed cells were stained with an anti-pRPA2 S4/S8 antibody. b Representative images of chromatin-bound pRPA2 S4/S8. Scale bar: 20 μm. c The intensity of chromatin-bound pRPA2 S4/S8 staining was quantified from ~20,000 cells. Error bars represent standard deviation of the mean (n = 3). Statistical analysis: t test; *p < 0.05. d U2OS cells expressing ATAD5AID were pre-treated with auxin and treated with 2 mM HU for 3 or 6 h. Chromatin-bound proteins were separated by SDS-PAGE and subjected for immunoblotting with indicated antibodies. e U2OS cells transfected with a combination of ATAD5 siRNA and a DNA vector expressing ATAD5-myc under the Noco-APH condition were treated with 2 mM HU for 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. f U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU or 1 μM CDC7 inhibitor (CDC7i, PHA-76941) for the indicated times. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. g HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min prior to treatment with 2 mM HU as indicated. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. h U2OS-TetOn-ATAD5 cells treated with doxycyclinfor 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected for immunoblotting. i, j U2OS cells transfected with siRNAs or treated with a RAD51 inhibitor (B02, 10, 20, 40 μM) at the time of release from aphidicolin under the Noco-APH condition were treated with 2 mM HU as indicated. Chromatin-bound proteins were fractionated and subjected for immunoblotting.
    U2os Cells Expressing Ostir1 9myc, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/u2os+cells+expressing+ostir1+9myc/pBabe+Blast+osTIR1-9Myc+(Plasmid+%2380073)/pm31844045-310-0-16
    Average 93 stars, based on 13 article reviews
    u2os cells expressing ostir1 9myc - by Bioz Stars, 2026-09
    93/100 stars

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    1) Product Images from "ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress."

    Article Title: ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

    Journal: Nature communications

    doi: 10.1038/s41467-019-13667-4

    Fig. 4 ATAD5 promotes generation of single-stranded DNA-associated breaks in response to replication stress. a U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 or 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. b, c U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 h before fixation. The fixed cells were stained with an anti-pRPA2 S4/S8 antibody. b Representative images of chromatin-bound pRPA2 S4/S8. Scale bar: 20 μm. c The intensity of chromatin-bound pRPA2 S4/S8 staining was quantified from ~20,000 cells. Error bars represent standard deviation of the mean (n = 3). Statistical analysis: t test; *p < 0.05. d U2OS cells expressing ATAD5AID were pre-treated with auxin and treated with 2 mM HU for 3 or 6 h. Chromatin-bound proteins were separated by SDS-PAGE and subjected for immunoblotting with indicated antibodies. e U2OS cells transfected with a combination of ATAD5 siRNA and a DNA vector expressing ATAD5-myc under the Noco-APH condition were treated with 2 mM HU for 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. f U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU or 1 μM CDC7 inhibitor (CDC7i, PHA-76941) for the indicated times. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. g HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min prior to treatment with 2 mM HU as indicated. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. h U2OS-TetOn-ATAD5 cells treated with doxycyclinfor 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected for immunoblotting. i, j U2OS cells transfected with siRNAs or treated with a RAD51 inhibitor (B02, 10, 20, 40 μM) at the time of release from aphidicolin under the Noco-APH condition were treated with 2 mM HU as indicated. Chromatin-bound proteins were fractionated and subjected for immunoblotting.
    Figure Legend Snippet: Fig. 4 ATAD5 promotes generation of single-stranded DNA-associated breaks in response to replication stress. a U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 or 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. b, c U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 h before fixation. The fixed cells were stained with an anti-pRPA2 S4/S8 antibody. b Representative images of chromatin-bound pRPA2 S4/S8. Scale bar: 20 μm. c The intensity of chromatin-bound pRPA2 S4/S8 staining was quantified from ~20,000 cells. Error bars represent standard deviation of the mean (n = 3). Statistical analysis: t test; *p < 0.05. d U2OS cells expressing ATAD5AID were pre-treated with auxin and treated with 2 mM HU for 3 or 6 h. Chromatin-bound proteins were separated by SDS-PAGE and subjected for immunoblotting with indicated antibodies. e U2OS cells transfected with a combination of ATAD5 siRNA and a DNA vector expressing ATAD5-myc under the Noco-APH condition were treated with 2 mM HU for 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. f U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU or 1 μM CDC7 inhibitor (CDC7i, PHA-76941) for the indicated times. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. g HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min prior to treatment with 2 mM HU as indicated. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. h U2OS-TetOn-ATAD5 cells treated with doxycyclinfor 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected for immunoblotting. i, j U2OS cells transfected with siRNAs or treated with a RAD51 inhibitor (B02, 10, 20, 40 μM) at the time of release from aphidicolin under the Noco-APH condition were treated with 2 mM HU as indicated. Chromatin-bound proteins were fractionated and subjected for immunoblotting.

    Techniques Used: Transfection, Western Blot, Staining, Standard Deviation, Expressing, SDS Page, Plasmid Preparation, Labeling

    Fig. 5 ATAD5 promotes generation of MUS81-mediated single-stranded DNA-associated breaks in response to replication stress. a, b U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 6 h before being collected for analysis by pulsed field gel electrophoresis. a Representative data from three independent experiments. Asterisk (*) indicates a DNA break. b DNA breaks were quantified and displayed (N = 4). c U2OS or HeLa cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 6 h before being collected for a neutral COMET assay. The tail moment was calculated from ~200 cells and plotted. d U2OS cells expressing ATAD5AID were pre-treated with auxin and treated with 2 mM HU for another 6 h before being collected for a neutral COMET assay. Two independent experiments were performed, and one representative result is displayed. e U2OS-TetOn-ATAD5 cells treated with doxycycline for 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h before collection for the neutral COMET assay. f U2OS cells transfected with siRNAs under the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected to immunoblotting. g U2OS cells transfected with a combination of ATAD5 and MUS81 siRNAs under the Noco-APH condition were treated with 2 mM HU for 6 h before collection for the neutral COMET assay. The tail moment was calculated from ~300 cells and plotted. h HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min, washed, and treated with 2 mM HU for the indicated times. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. b–e, g Error bars represent standard deviation of the mean. Statistical analysis: two-tailed Student’s t test; *p < 0.05, **p < 0.005, ***p < 0.001, and ****p < 0.0001.
    Figure Legend Snippet: Fig. 5 ATAD5 promotes generation of MUS81-mediated single-stranded DNA-associated breaks in response to replication stress. a, b U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 6 h before being collected for analysis by pulsed field gel electrophoresis. a Representative data from three independent experiments. Asterisk (*) indicates a DNA break. b DNA breaks were quantified and displayed (N = 4). c U2OS or HeLa cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 6 h before being collected for a neutral COMET assay. The tail moment was calculated from ~200 cells and plotted. d U2OS cells expressing ATAD5AID were pre-treated with auxin and treated with 2 mM HU for another 6 h before being collected for a neutral COMET assay. Two independent experiments were performed, and one representative result is displayed. e U2OS-TetOn-ATAD5 cells treated with doxycycline for 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h before collection for the neutral COMET assay. f U2OS cells transfected with siRNAs under the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected to immunoblotting. g U2OS cells transfected with a combination of ATAD5 and MUS81 siRNAs under the Noco-APH condition were treated with 2 mM HU for 6 h before collection for the neutral COMET assay. The tail moment was calculated from ~300 cells and plotted. h HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min, washed, and treated with 2 mM HU for the indicated times. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. b–e, g Error bars represent standard deviation of the mean. Statistical analysis: two-tailed Student’s t test; *p < 0.05, **p < 0.005, ***p < 0.001, and ****p < 0.0001.

    Techniques Used: Transfection, Nucleic Acid Electrophoresis, Neutral Comet Assay, Expressing, Western Blot, Labeling, SDS Page, Standard Deviation, Two Tailed Test

    Related Articles

    Expressing:

    Article Title: ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.
    Article Snippet: Human embryonic kidney (HEK) 293T (ATCC® CRL-3216TM), HeLa (ATCC® CCL-2TM), and U2OS (ATCC® HTB-96TM) cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (GE Healthcare, Little Chalfont, UK), 100 U/mL penicillin G (Life Technologies, Carlsbad, CA), and 100 μg/mL streptomycin (Life Technologies). .. U2OS cells expressing osTIR1-9Myc were generated by retroviral infection of pBabe Blast osTIR1-9Myc from Andrew Holland (Addgene, plasmid # 80073). .. To generate ATAD5AID cell line, U2OS-osTIR19Myc cells were co-transfected with CRISPR/Cas9, single guide RNA (see Supplementary Table 2 for the sequence information) and donor plasmids by nucleofection (Lonza).

    Generated:

    Article Title: ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.
    Article Snippet: Human embryonic kidney (HEK) 293T (ATCC® CRL-3216TM), HeLa (ATCC® CCL-2TM), and U2OS (ATCC® HTB-96TM) cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (GE Healthcare, Little Chalfont, UK), 100 U/mL penicillin G (Life Technologies, Carlsbad, CA), and 100 μg/mL streptomycin (Life Technologies). .. U2OS cells expressing osTIR1-9Myc were generated by retroviral infection of pBabe Blast osTIR1-9Myc from Andrew Holland (Addgene, plasmid # 80073). .. To generate ATAD5AID cell line, U2OS-osTIR19Myc cells were co-transfected with CRISPR/Cas9, single guide RNA (see Supplementary Table 2 for the sequence information) and donor plasmids by nucleofection (Lonza).

    Retroviral:

    Article Title: ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.
    Article Snippet: Human embryonic kidney (HEK) 293T (ATCC® CRL-3216TM), HeLa (ATCC® CCL-2TM), and U2OS (ATCC® HTB-96TM) cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (GE Healthcare, Little Chalfont, UK), 100 U/mL penicillin G (Life Technologies, Carlsbad, CA), and 100 μg/mL streptomycin (Life Technologies). .. U2OS cells expressing osTIR1-9Myc were generated by retroviral infection of pBabe Blast osTIR1-9Myc from Andrew Holland (Addgene, plasmid # 80073). .. To generate ATAD5AID cell line, U2OS-osTIR19Myc cells were co-transfected with CRISPR/Cas9, single guide RNA (see Supplementary Table 2 for the sequence information) and donor plasmids by nucleofection (Lonza).

    Infection:

    Article Title: ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.
    Article Snippet: Human embryonic kidney (HEK) 293T (ATCC® CRL-3216TM), HeLa (ATCC® CCL-2TM), and U2OS (ATCC® HTB-96TM) cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (GE Healthcare, Little Chalfont, UK), 100 U/mL penicillin G (Life Technologies, Carlsbad, CA), and 100 μg/mL streptomycin (Life Technologies). .. U2OS cells expressing osTIR1-9Myc were generated by retroviral infection of pBabe Blast osTIR1-9Myc from Andrew Holland (Addgene, plasmid # 80073). .. To generate ATAD5AID cell line, U2OS-osTIR19Myc cells were co-transfected with CRISPR/Cas9, single guide RNA (see Supplementary Table 2 for the sequence information) and donor plasmids by nucleofection (Lonza).

    Plasmid Preparation:

    Article Title: ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.
    Article Snippet: Human embryonic kidney (HEK) 293T (ATCC® CRL-3216TM), HeLa (ATCC® CCL-2TM), and U2OS (ATCC® HTB-96TM) cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (GE Healthcare, Little Chalfont, UK), 100 U/mL penicillin G (Life Technologies, Carlsbad, CA), and 100 μg/mL streptomycin (Life Technologies). .. U2OS cells expressing osTIR1-9Myc were generated by retroviral infection of pBabe Blast osTIR1-9Myc from Andrew Holland (Addgene, plasmid # 80073). .. To generate ATAD5AID cell line, U2OS-osTIR19Myc cells were co-transfected with CRISPR/Cas9, single guide RNA (see Supplementary Table 2 for the sequence information) and donor plasmids by nucleofection (Lonza).



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    u2os cells expressing ostir1 9myc  (Addgene inc)
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    Addgene inc u2os cells expressing ostir1 9myc
    Fig. 4 ATAD5 promotes generation of single-stranded DNA-associated breaks in response to replication stress. a <t>U2OS</t> cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 or 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. b, c U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 h before fixation. The fixed cells were stained with an anti-pRPA2 S4/S8 antibody. b Representative images of chromatin-bound pRPA2 S4/S8. Scale bar: 20 μm. c The intensity of chromatin-bound pRPA2 S4/S8 staining was quantified from ~20,000 cells. Error bars represent standard deviation of the mean (n = 3). Statistical analysis: t test; *p < 0.05. d U2OS cells expressing ATAD5AID were pre-treated with auxin and treated with 2 mM HU for 3 or 6 h. Chromatin-bound proteins were separated by SDS-PAGE and subjected for immunoblotting with indicated antibodies. e U2OS cells transfected with a combination of ATAD5 siRNA and a DNA vector expressing ATAD5-myc under the Noco-APH condition were treated with 2 mM HU for 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. f U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU or 1 μM CDC7 inhibitor (CDC7i, PHA-76941) for the indicated times. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. g HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min prior to treatment with 2 mM HU as indicated. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. h U2OS-TetOn-ATAD5 cells treated with doxycyclinfor 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected for immunoblotting. i, j U2OS cells transfected with siRNAs or treated with a RAD51 inhibitor (B02, 10, 20, 40 μM) at the time of release from aphidicolin under the Noco-APH condition were treated with 2 mM HU as indicated. Chromatin-bound proteins were fractionated and subjected for immunoblotting.
    U2os Cells Expressing Ostir1 9myc, supplied by Addgene 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/product/u2os+cells+expressing+ostir1+9myc/pBabe+Blast+osTIR1-9Myc+(Plasmid+%2380073)/pm31844045-310-0-16
    Average 93 stars, based on 1 article reviews
    u2os cells expressing ostir1 9myc - by Bioz Stars, 2026-09
    93/100 stars
    		  Buy from Supplier

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    Fig. 4 ATAD5 promotes generation of single-stranded DNA-associated breaks in response to replication stress. a U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 or 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. b, c U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 h before fixation. The fixed cells were stained with an anti-pRPA2 S4/S8 antibody. b Representative images of chromatin-bound pRPA2 S4/S8. Scale bar: 20 μm. c The intensity of chromatin-bound pRPA2 S4/S8 staining was quantified from ~20,000 cells. Error bars represent standard deviation of the mean (n = 3). Statistical analysis: t test; *p < 0.05. d U2OS cells expressing ATAD5AID were pre-treated with auxin and treated with 2 mM HU for 3 or 6 h. Chromatin-bound proteins were separated by SDS-PAGE and subjected for immunoblotting with indicated antibodies. e U2OS cells transfected with a combination of ATAD5 siRNA and a DNA vector expressing ATAD5-myc under the Noco-APH condition were treated with 2 mM HU for 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. f U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU or 1 μM CDC7 inhibitor (CDC7i, PHA-76941) for the indicated times. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. g HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min prior to treatment with 2 mM HU as indicated. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. h U2OS-TetOn-ATAD5 cells treated with doxycyclinfor 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected for immunoblotting. i, j U2OS cells transfected with siRNAs or treated with a RAD51 inhibitor (B02, 10, 20, 40 μM) at the time of release from aphidicolin under the Noco-APH condition were treated with 2 mM HU as indicated. Chromatin-bound proteins were fractionated and subjected for immunoblotting.

    Journal: Nature communications

    Article Title: ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

    doi: 10.1038/s41467-019-13667-4

    Figure Lengend Snippet: Fig. 4 ATAD5 promotes generation of single-stranded DNA-associated breaks in response to replication stress. a U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 or 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. b, c U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 h before fixation. The fixed cells were stained with an anti-pRPA2 S4/S8 antibody. b Representative images of chromatin-bound pRPA2 S4/S8. Scale bar: 20 μm. c The intensity of chromatin-bound pRPA2 S4/S8 staining was quantified from ~20,000 cells. Error bars represent standard deviation of the mean (n = 3). Statistical analysis: t test; *p < 0.05. d U2OS cells expressing ATAD5AID were pre-treated with auxin and treated with 2 mM HU for 3 or 6 h. Chromatin-bound proteins were separated by SDS-PAGE and subjected for immunoblotting with indicated antibodies. e U2OS cells transfected with a combination of ATAD5 siRNA and a DNA vector expressing ATAD5-myc under the Noco-APH condition were treated with 2 mM HU for 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. f U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU or 1 μM CDC7 inhibitor (CDC7i, PHA-76941) for the indicated times. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. g HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min prior to treatment with 2 mM HU as indicated. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. h U2OS-TetOn-ATAD5 cells treated with doxycyclinfor 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected for immunoblotting. i, j U2OS cells transfected with siRNAs or treated with a RAD51 inhibitor (B02, 10, 20, 40 μM) at the time of release from aphidicolin under the Noco-APH condition were treated with 2 mM HU as indicated. Chromatin-bound proteins were fractionated and subjected for immunoblotting.

    Article Snippet: U2OS cells expressing osTIR1-9Myc were generated by retroviral infection of pBabe Blast osTIR1-9Myc from Andrew Holland (Addgene, plasmid # 80073).

    Techniques: Transfection, Western Blot, Staining, Standard Deviation, Expressing, SDS Page, Plasmid Preparation, Labeling

    Fig. 5 ATAD5 promotes generation of MUS81-mediated single-stranded DNA-associated breaks in response to replication stress. a, b U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 6 h before being collected for analysis by pulsed field gel electrophoresis. a Representative data from three independent experiments. Asterisk (*) indicates a DNA break. b DNA breaks were quantified and displayed (N = 4). c U2OS or HeLa cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 6 h before being collected for a neutral COMET assay. The tail moment was calculated from ~200 cells and plotted. d U2OS cells expressing ATAD5AID were pre-treated with auxin and treated with 2 mM HU for another 6 h before being collected for a neutral COMET assay. Two independent experiments were performed, and one representative result is displayed. e U2OS-TetOn-ATAD5 cells treated with doxycycline for 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h before collection for the neutral COMET assay. f U2OS cells transfected with siRNAs under the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected to immunoblotting. g U2OS cells transfected with a combination of ATAD5 and MUS81 siRNAs under the Noco-APH condition were treated with 2 mM HU for 6 h before collection for the neutral COMET assay. The tail moment was calculated from ~300 cells and plotted. h HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min, washed, and treated with 2 mM HU for the indicated times. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. b–e, g Error bars represent standard deviation of the mean. Statistical analysis: two-tailed Student’s t test; *p < 0.05, **p < 0.005, ***p < 0.001, and ****p < 0.0001.

    Journal: Nature communications

    Article Title: ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

    doi: 10.1038/s41467-019-13667-4

    Figure Lengend Snippet: Fig. 5 ATAD5 promotes generation of MUS81-mediated single-stranded DNA-associated breaks in response to replication stress. a, b U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 6 h before being collected for analysis by pulsed field gel electrophoresis. a Representative data from three independent experiments. Asterisk (*) indicates a DNA break. b DNA breaks were quantified and displayed (N = 4). c U2OS or HeLa cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 6 h before being collected for a neutral COMET assay. The tail moment was calculated from ~200 cells and plotted. d U2OS cells expressing ATAD5AID were pre-treated with auxin and treated with 2 mM HU for another 6 h before being collected for a neutral COMET assay. Two independent experiments were performed, and one representative result is displayed. e U2OS-TetOn-ATAD5 cells treated with doxycycline for 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h before collection for the neutral COMET assay. f U2OS cells transfected with siRNAs under the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected to immunoblotting. g U2OS cells transfected with a combination of ATAD5 and MUS81 siRNAs under the Noco-APH condition were treated with 2 mM HU for 6 h before collection for the neutral COMET assay. The tail moment was calculated from ~300 cells and plotted. h HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min, washed, and treated with 2 mM HU for the indicated times. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. b–e, g Error bars represent standard deviation of the mean. Statistical analysis: two-tailed Student’s t test; *p < 0.05, **p < 0.005, ***p < 0.001, and ****p < 0.0001.

    Article Snippet: U2OS cells expressing osTIR1-9Myc were generated by retroviral infection of pBabe Blast osTIR1-9Myc from Andrew Holland (Addgene, plasmid # 80073).

    Techniques: Transfection, Nucleic Acid Electrophoresis, Neutral Comet Assay, Expressing, Western Blot, Labeling, SDS Page, Standard Deviation, Two Tailed Test