Journal: Cell reports
Article Title: Identification of the main barriers to Ku accumulation in chromatin
doi: 10.1016/j.celrep.2024.114538
Figure Lengend Snippet: (A) Immunoblot of U2OS cells that are wild type (WT) or knocked out for the indicated genes. See for the IR sensitivity analysis of each of these cells. (B) WT, PKcs-KO, LIG4-KO, or LIG4/PKcs-KO received 5 Gy of IR before being post-incubated 5 or 60 min and processed for Ku foci imaging. Representative pictures are shown on the left. Ku foci intensity was measured and normalized to the average Ku foci intensity measured after 5 Gy of IR in WT U2OS to compute the fold change in Ku foci intensity in each condition, depicted on the graph on the right. (C) WT or PKcs-KO U2OS cells received 5 Gy of IR and were post-incubated for 60 min before being processed for Ku foci imaging. Cells were pre-treated with nedisertib (PKi) for 1 h before treatment where indicated. Fold change in Ku foci intensity in each condition is displayed. (D) PKcs-KO or LIG4-KO U2OS cells received 5 Gy of IR and were post-incubated for the indicated time with or without NEDi before being processed for Ku foci imaging. Representative pictures are shown on the left, while the ratio between the changes in Ku foci intensity with NEDi versus without NEDi (DMSO) are shown in the graph on the right. The graph depicting the fold change in Ku foci intensity in each condition corresponds to . (E) WT, PKcs-KO, LIG4-KO, or LIG4/PKcs-KO U2OS cells received 5 Gy of IR and were post-incubated for 5 or 60 min with or without NEDi before being processed for Ku foci imaging. Representative pictures are shown on the left, while the ratio between the change in Ku foci intensity with NEDi versus without NEDi (DMSO) is plotted in the graph on the right. The graph in depicts the fold change in Ku foci intensity in each condition. (F) WT, PKcs-KO, or LIG4-KO U2OS cells incubated with 3 μM NU7441 (PKi) were treated with 3 nM Cali for 1 h with or without NEDi before being collected and processed to separate the chromatin fraction from the soluble fraction, which were both analyzed by immunoblotting. SAF-A and nucleolin were used as loading controls for the chromatin and the soluble fraction, respectively. (G) Control (immunoglobulin G [IgG]) or anti-Ku immunoprecipitation was performed from the soluble fractions of PKcs-KO U2OS to monitor Ku ubiquitination in response to Cali with or without NEDi. For all panels, error bars represent SD from the means of n ≥ 3 independent experiments. p values are as follow: ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Scale bars, 5 μm.
Article Snippet: NU7441 (studies in human) , Tocris , Cat# 3712.
Techniques: Western Blot, Incubation, Imaging, Control, Immunoprecipitation
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![(A) Immunoblot of U2OS cells that are wild type (WT) or knocked out for the indicated genes. See for the IR sensitivity analysis of each of these cells. (B) WT, PKcs-KO, LIG4-KO, or LIG4/PKcs-KO received 5 Gy of IR before being post-incubated 5 or 60 min and processed for Ku foci imaging. Representative pictures are shown on the left. Ku foci intensity was measured and normalized to the average Ku foci intensity measured after 5 Gy of IR in WT U2OS to compute the fold change in Ku foci intensity in each condition, depicted on the graph on the right. (C) WT or PKcs-KO U2OS cells received 5 Gy of IR and were post-incubated for 60 min before being processed for Ku foci imaging. Cells were pre-treated with nedisertib (PKi) for 1 h before treatment where indicated. Fold change in Ku foci intensity in each condition is displayed. (D) PKcs-KO or LIG4-KO U2OS cells received 5 Gy of IR and were post-incubated for the indicated time with or without NEDi before being processed for Ku foci imaging. Representative pictures are shown on the left, while the ratio between the changes in Ku foci intensity with NEDi versus without NEDi (DMSO) are shown in the graph on the right. The graph depicting the fold change in Ku foci intensity in each condition corresponds to . (E) WT, PKcs-KO, LIG4-KO, or LIG4/PKcs-KO U2OS cells received 5 Gy of IR and were post-incubated for 5 or 60 min with or without NEDi before being processed for Ku foci imaging. Representative pictures are shown on the left, while the ratio between the change in Ku foci intensity with NEDi versus without NEDi (DMSO) is plotted in the graph on the right. The graph in depicts the fold change in Ku foci intensity in each condition. (F) WT, PKcs-KO, or LIG4-KO U2OS cells incubated with 3 μM <t>NU7441</t> (PKi) were treated with 3 nM Cali for 1 h with or without NEDi before being collected and processed to separate the chromatin fraction from the soluble fraction, which were both analyzed by immunoblotting. SAF-A and nucleolin were used as loading controls for the chromatin and the soluble fraction, respectively. (G) Control (immunoglobulin G [IgG]) or anti-Ku immunoprecipitation was performed from the soluble fractions of PKcs-KO U2OS to monitor Ku ubiquitination in response to Cali with or without NEDi. For all panels, error bars represent SD from the means of n ≥ 3 independent experiments. p values are as follow: ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Scale bars, 5 μm.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1529/pmc11411529/pmc11411529__nihms-2019515-f0002.jpg)
