Fig. 5. Inhibiting PARG triggers TOP1-DPC deubiquitylation by USP7.

a His-tag pulldown-HPLC-MS/MS showing that His-TOP1 interacted USP7 under unperturbed condition. After transfection of 6×His-tagged TOP1 expression plasmid or empty vector control (pTrex), HCT116 cells were subjected to His-tag pulldown using Ni-NTA agarose, followed by HPLC-MS. b In vitro assay showing that USP7 reversed TOP1 ubiquitylation. Recombinant TOP1 was subjected to ubiquitylation with ubiquitin, Ube1 (E1), Ubc5Hα (E2), and RNF4 (E3) for 30 min, followed by termination with EDTA and incubation with increasing concentrations of recombinant USP7 for another 30 min. Samples were Western blotted with α-Ub antibody. c His-tag pulldown assay showing that PARGi enhanced TOP1-USP7 interaction. Following transfection of 6×His-tagged TOP1 expression plasmid and FLAG-USP7 expression plasmid, HEK293 cells were treated as indicated. His-tag pulldown was performed with Ni-NTA agarose under native conditions. Western blotting was performed with the indicated antibodies. d PLA assay showing TOP1-USP7 interaction in PARGi-treated cells. Following transfection of 6×His-tagged TOP1 expression plasmid and FLAG-USP7 expression plasmid, HEK293 cells were treated as indicated. PLA assays were performed using rabbit α-His-tag antibody and mouse α-FLAG tag antibody. The scale bar represents 10 μm. e Inhibiting USP7 restored TOP1-DPC ubiquitylation in the presence but not in the absence of PARGi. Upper panel: HEK293 cells were treated as indicated: CPT (20 µM, 1 h), CPT + FLAG-USP7 transfection, CPT + USP7i (10 µM, 1 h pre-treatment), CPT + PARGi (10 µM, 1 h pre-treatment), CPT + PARGi + FLAG-USP7 transfection, CPT + PARGi + USP7i. Following treatments, cells were subjected to the modified RADAR assay for detection of TOP1-DPCs and their ubiquitylation using α-TOP1 and α-Ub antibodies. Lower panel: densitometric quantitation of ubiquitylated TOP1-DPC signals generated from triplicate experiments including representative blots shown in (c) using ImageJ. n = 3 independent experiments. Data are presented as mean values +/− standard deviation (SD). P value was calculated by paired Student’s t-test (two-tailed distribution). *: p = . NS not significant. f Inhibiting USP7 did not impact the induction of γH2AX upon exposure to CPT. U2OS cells were synchronized in the S phase by double thymidine and treated with CPT (1 µM) in the absence of presence of USP7i (10 µM, 1 h pre-treatment) and collected for IF by iSIM using an anti-γH2AX antibody. The scale bar represents 10 μm.