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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.
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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