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Regulated phosphorylation of WRN at S1141 is involved in RAD51 localization. ( A ) Cells stably expressing Flag-tagged WRN mutants were treated with CPT and allowed to recover for different time points as indicated. RAD51-foci staining was then analysed by IF. The graph shows the percentage of RAD51-foci positive cells measured from two independent experiments ( n = 200, each biological replicate). Data are presented as mea ± SE. Representative images of RAD51 staining in response to treatment are shown. ( B ) Analysis of ssDNA/RAD51 interaction by in <t>situ</t> <t>PLA.</t> Cells were treated with CPT and allowed to recover for 4 h, then subjected to PLA using anti-IdU and anti-RAD51 antibodies. The panels show representative PLA images showing association of ssDNA with RAD51 (scale bar: 10 μm). The dot plot shows the PLA spots per PLA positive cells. At least 100 nuclei were analysed for each experimental point ( n = 2). Values are presented as mean ± SE. Statistical analysis was performed by the ANOVA test (**** P < 0.0001, ** P < 0.01, each biological replicate). ( C ) The graph shows the number of PLA positive cells. At least 100 nuclei were analysed for each experimental point. Representative images from neutral Comet assay are shown.
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Millipore pla
Regulated phosphorylation of WRN at S1141 is involved in RAD51 localization. ( A ) Cells stably expressing Flag-tagged WRN mutants were treated with CPT and allowed to recover for different time points as indicated. RAD51-foci staining was then analysed by IF. The graph shows the percentage of RAD51-foci positive cells measured from two independent experiments ( n = 200, each biological replicate). Data are presented as mea ± SE. Representative images of RAD51 staining in response to treatment are shown. ( B ) Analysis of ssDNA/RAD51 interaction by in <t>situ</t> <t>PLA.</t> Cells were treated with CPT and allowed to recover for 4 h, then subjected to PLA using anti-IdU and anti-RAD51 antibodies. The panels show representative PLA images showing association of ssDNA with RAD51 (scale bar: 10 μm). The dot plot shows the PLA spots per PLA positive cells. At least 100 nuclei were analysed for each experimental point ( n = 2). Values are presented as mean ± SE. Statistical analysis was performed by the ANOVA test (**** P < 0.0001, ** P < 0.01, each biological replicate). ( C ) The graph shows the number of PLA positive cells. At least 100 nuclei were analysed for each experimental point. Representative images from neutral Comet assay are shown.
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Regulated phosphorylation of WRN at S1141 is involved in RAD51 localization. ( A ) Cells stably expressing Flag-tagged WRN mutants were treated with CPT and allowed to recover for different time points as indicated. RAD51-foci staining was then analysed by IF. The graph shows the percentage of RAD51-foci positive cells measured from two independent experiments ( n = 200, each biological replicate). Data are presented as mea ± SE. Representative images of RAD51 staining in response to treatment are shown. ( B ) Analysis of ssDNA/RAD51 interaction by in situ PLA. Cells were treated with CPT and allowed to recover for 4 h, then subjected to PLA using anti-IdU and anti-RAD51 antibodies. The panels show representative PLA images showing association of ssDNA with RAD51 (scale bar: 10 μm). The dot plot shows the PLA spots per PLA positive cells. At least 100 nuclei were analysed for each experimental point ( n = 2). Values are presented as mean ± SE. Statistical analysis was performed by the ANOVA test (**** P < 0.0001, ** P < 0.01, each biological replicate). ( C ) The graph shows the number of PLA positive cells. At least 100 nuclei were analysed for each experimental point. Representative images from neutral Comet assay are shown.

Journal: Nucleic Acids Research

Article Title: Switch-like phosphorylation of WRN integrates end-resection with RAD51 metabolism at collapsed replication forks

doi: 10.1093/nar/gkae807

Figure Lengend Snippet: Regulated phosphorylation of WRN at S1141 is involved in RAD51 localization. ( A ) Cells stably expressing Flag-tagged WRN mutants were treated with CPT and allowed to recover for different time points as indicated. RAD51-foci staining was then analysed by IF. The graph shows the percentage of RAD51-foci positive cells measured from two independent experiments ( n = 200, each biological replicate). Data are presented as mea ± SE. Representative images of RAD51 staining in response to treatment are shown. ( B ) Analysis of ssDNA/RAD51 interaction by in situ PLA. Cells were treated with CPT and allowed to recover for 4 h, then subjected to PLA using anti-IdU and anti-RAD51 antibodies. The panels show representative PLA images showing association of ssDNA with RAD51 (scale bar: 10 μm). The dot plot shows the PLA spots per PLA positive cells. At least 100 nuclei were analysed for each experimental point ( n = 2). Values are presented as mean ± SE. Statistical analysis was performed by the ANOVA test (**** P < 0.0001, ** P < 0.01, each biological replicate). ( C ) The graph shows the number of PLA positive cells. At least 100 nuclei were analysed for each experimental point. Representative images from neutral Comet assay are shown.

Article Snippet: In situ PLA (Merck) was performed according to the manufacturer’s instruction.

Techniques: Stable Transfection, Expressing, Staining, In Situ, Neutral Comet Assay