Figure 6
Figure 6. p30 induces a switch from the HR to the NHEJ DNA-repair pathway during the S phase. (A) p30 hampers the recruitment of the MRN complex on naturally occurring breaks. HeLa control or p30-expressing cells were stained with anti-MRE11 and anti p-H2AX antibodies to reveal natural breaks. (B) p30 favors NHEJ DNA repair. Representation of the in vivo NHEJ assay based on the EJ5-GFP reporter vector. HeLa cells were transfected with EJ5-GFP and pSceI expression vector with or without p30 expression vector. The percentage of GFP+ cells was estimated by FACS and is represented by the histograms of 3 independent experiments with standard deviations. (C) p30 favors unfaithful DNA repair. HeLa cells infected with mock lentivirus or p30-expressing lentivirus particles were treated or not with the DNA-PK inhibitor Nu7026 (20μM), a specific inhibitor of the NHEJ-repair pathway. Cells were γ-irradiated and the time course of the DNA-repair rate at 0, 2, 8, and 24 hours was performed by immunofluorescence staining with anti–p-H2AX antibody showing the number of breaks in each time condition. (D) DSB foci were counted in at least 30 cells collected by z-stack acquisition at 0 and 24 hours of each condition. Results represent average values. ***P < .001. (E) NHEJ DNA repair is increased in p30-expressing cells. HeLa cells transfected with GFP-p30 were synchronized with hydroxyurea and released for 7 hours to have the majority of cells in the S phase. The cells were then irradiated (10 Gy) and 2 hours later stained with anti-cyclin A (a marker of the S phase) or anti–DNA-PK (a marker of NHEJ) antibodies, followed by Alexa Fluor 568 (red) or Alexa Fluor 647 (blue)–conjugated secondary antibodies, respectively. (F) p30 favors a switch from the HR to the NHEJ DNA-repair. HeLa cells transfected with GFP-p30 were synchronized with hydroxyurea and released for 7 hours to have the majority of cells in the S phase. Cells were irradiated (10 Gy) and 2 hours later stained with anti–p-H2AX (DSB) and either Rad50 (HR-specific) or DNA-PK (NHEJ-specific) antibodies.

p30 induces a switch from the HR to the NHEJ DNA-repair pathway during the S phase. (A) p30 hampers the recruitment of the MRN complex on naturally occurring breaks. HeLa control or p30-expressing cells were stained with anti-MRE11 and anti p-H2AX antibodies to reveal natural breaks. (B) p30 favors NHEJ DNA repair. Representation of the in vivo NHEJ assay based on the EJ5-GFP reporter vector. HeLa cells were transfected with EJ5-GFP and pSceI expression vector with or without p30 expression vector. The percentage of GFP+ cells was estimated by FACS and is represented by the histograms of 3 independent experiments with standard deviations. (C) p30 favors unfaithful DNA repair. HeLa cells infected with mock lentivirus or p30-expressing lentivirus particles were treated or not with the DNA-PK inhibitor Nu7026 (20μM), a specific inhibitor of the NHEJ-repair pathway. Cells were γ-irradiated and the time course of the DNA-repair rate at 0, 2, 8, and 24 hours was performed by immunofluorescence staining with anti–p-H2AX antibody showing the number of breaks in each time condition. (D) DSB foci were counted in at least 30 cells collected by z-stack acquisition at 0 and 24 hours of each condition. Results represent average values. ***P < .001. (E) NHEJ DNA repair is increased in p30-expressing cells. HeLa cells transfected with GFP-p30 were synchronized with hydroxyurea and released for 7 hours to have the majority of cells in the S phase. The cells were then irradiated (10 Gy) and 2 hours later stained with anti-cyclin A (a marker of the S phase) or anti–DNA-PK (a marker of NHEJ) antibodies, followed by Alexa Fluor 568 (red) or Alexa Fluor 647 (blue)–conjugated secondary antibodies, respectively. (F) p30 favors a switch from the HR to the NHEJ DNA-repair. HeLa cells transfected with GFP-p30 were synchronized with hydroxyurea and released for 7 hours to have the majority of cells in the S phase. Cells were irradiated (10 Gy) and 2 hours later stained with anti–p-H2AX (DSB) and either Rad50 (HR-specific) or DNA-PK (NHEJ-specific) antibodies.

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