Fig. 5: DNA damage generates a ROS signal that promotes ATM-dependent stabilization of PARP1 via USP10. | Oncogene

Fig. 5: DNA damage generates a ROS signal that promotes ATM-dependent stabilization of PARP1 via USP10.

From: The deubiquitination-PARylation positive feedback loop of the USP10-PARP1 axis promotes DNA damage repair and affects therapeutic efficacy of PARP1 inhibitor

Fig. 5

A Western blot analysis of PARP1 protein levels in HEK293 cells treated with HU (5 mM) for the indicated time. B Western blot analysis of PARP1 in MCF7 cells pretreated with or without NAC (3 mM) and then treated with or without HU (10 mM) for 4 h. PARP1 ubiquitination level was determined in HEK293 cells treated with or without H2O2 (500 μM) for 5 h (C), treated with or without HU (10 mM) for 7 h (D). Lysates were immunoprecipitated with IgG control or anti-PARP1 antibody, followed by immunoblotting with anti-ubiquitin antibody. E HEK293 cells pretreated with or without NAC (3 mM) and then treated with or without HU (5 mM) for 4 h. Lysates were immunoprecipitated with anti-PARP1 antibody, followed by immunoblotting with anti-ubiquitin antibody. F HEK293 cells transfected with Flag-USP10 treated with or without H2O2 (500 μM) for 4 h. Lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-ATM and anti-PARP1 antibody. G, H HEK293 cells transfected with Flag-USP10 pretreated with or without NAC (3 mM) and then treated with or without HU (15 mM) for 6 h. Lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-ATM antibody and anti-PARP1 antibody. I Immunofluorescence analysis of USP10 and PARP1 in HEK293 cells treated with or without H2O2 (100 μM) for 1 h. Cells were stained with anti-USP10, anti-PARP1, and DAPI. Scale bar, 10 μm. Pearson’s coefficient was calculated to quantify the co-localization of USP10 and PARP1. Data are presented as mean ± SEM from six independent experiments, ***P < 0.001 (Student’s t test). J Proximity ligation assay (PLA) passes were performed to determine the interaction between PARP1 and USP10. Red indicates the presence of interaction, and blue (DAPI) indicates nuclear staining. Scale bar, 10 μm. K H1299 control and shRNA ATM knockdown cells were treated with or without H2O2 (500 μM) for 1.5 h, and lysates were immunoblotted with the indicated antibodies. L H1299 control and shRNA ATM knockdown cells were treated with or without HU (20 mM) for 4 h. Lysates were immunoprecipitated with anti-PARP1 antibody, followed by immunoblotting with anti-ubiquitin antibody. M H1299 control and shRNA ATM knockdown cells were transfected with Flag-USP10 plasmids for 48 h, and then treated with or without H2O2 (500 μM) for 4 h. Lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-phospho-Ser/Thr antibody. N Analysis of PARP1 ubiquitination in HEK293 cells transfected with Flag-USP10 wild-type or Flag-USP10 T42A/S337A plasmids, treated with or without H2O2 (500 μM) for 4 h. Lysates were immunoprecipitated with anti-PARP1 antibody, followed by immunoblotting with anti-ubiquitin antibody. O Western blot analysis of PARP1 in HEK293 cells pretreated with or without Spautin-1 (20 μM) for 24 h and then treated with or without HU (10 mM) for 6 h. P PARP1 ubiquitination in MCF7 cells pretreated with or without Spautin-1 (30 μM) for 24 h and then treated with or without HU (30 mM) for 7 h. Lysates were immunoprecipitated with anti-PARP1 antibody, followed by immunoblotting with anti-ubiquitin antibody.

Back to article page