Figure 1:

USP22 is necessary for efficient HR of DSBs. a. H1299 cells treated with DNA damaging drugs Cisplatin or Camptothecin and fixed with indicated antibodies. (right) Western blot of control versus USP22 siRNA KD of H1299 cells after 48-hour treatment. β-Actin is used as a loading control in the input. Numbers to the left of western blots indicate molecular weight. b. Rad51 and H2A.X foci count per cell for experiment 1a. 120 cells were analyzed for every condition (i.e. 120 cells were analyzed for Rad51 control siRNA, 120 cells were analyzed for Rad51 USP22 siRNA etc.). Experiment was done in triplicate c. FACS analysis results of U2OS cells after USP22 or control siRNA KD and SceI endonuclease cleavage of truncated GFP gene³⁰. EJ5=NHEJ, HDR=Homology-directed repair, and SSA=Single strand annealing. Experiment was performed in triplicate. d. Western blot for experiment 1c with indicated antibodies for indicated cell line noted below. β-Actin is used as a loading control and molecular weight markers are on the left. For all statistical analysis a student’s two-tailed t-test was used to establish p-value. e. Western blot showing gamma-H2A.X is upregulated upon DNA damage in U2OS^FokI in the presence of a combined USP22/PALB2 knockdown. Cells were treated with siRNA for 48 hours then FokI treatment was performed for 6 hours before cells were harvested. Numbers to left of western blots indicate molecular weight.