Fig 2: USP7 and VCPFAF1 functions in DNA replication converge into the SUMOylation pathway(A and B) Immunofluorescence analysis of chromatin-bound VCP (green) and SUMO2/3 (red) (A) or SUMO2/3 (green) and MCM3 (red).(B) Levels in U2OS cells that were either untreated (CONTROL) or after treatment with 50 µM USP7i for 4 h. DNA was stained with DAPI (blue). The overlay for the different staining is also shown. Scale bar, 10 µm. The intensity of the individual stainings was quantified along the line indicated in the figure to assess the co-localization of the analyzed proteins (right).(C and D) HeLa cells were transfected with a non-specific siRNA (Con, black) or siRNA against FAF1 (FAF1, orange), and the levels of SUMO2/3 (C) or VCP (D) on chromatin were measured by high-throughput microscopy 48 h after transfection. Cells were treated with DMSO (Control) or 25 µM P22077 (USP7i) for the indicated times.(E and F) Representative images taken at 4 h of treatment are shown. The experiment was repeated 3 times, and one representative result is shown.(G) Western blot analysis of the inputs and eluates of an in vitro pull-down using GFP and SMO-1-GFP as bait. UBXN-3 or a deletion variant of the N-terminal UBA (?UBA) domain were used along with increasing amounts of tetra-Ubiquitin chains linked via Lysine 48 (4Ub(K48)). Note, that detection of Ubiquitin revealed cross-reactivity of the antibody with GFP. SMO-1-GFP-bound UBXN-3 can efficiently recover 4Ub(K48) without affecting UBXN-3 binding to SMO-1. UBXN-3(?UBA) interacts with SMO-1-GFP, while it is deficient in ubiquitin binding.(H) Schematic representation of quantitative analysis of UBXN-3 (green) and SMO-1 (red) spots in C. elegans embryonic nuclei. Raw images were masked for embryonic tissue and nuclei, before nuclear spots were defined for subsequent analysis. Images show a representative of npl-4-depleted embryos, immune labeled with UBXN-3 and SMO-1 antibodies.(I) Quantification of co-localized UBXN-3 (green) and SMO-1 (red) spot volume in embryos depleted for indicated gene products relative to green or red total spot volume, respectively. Whisker plots of the 10–90th percentile are shown, and statistical significance was interrogated using one-way ANOVA Sidak’s multiple-comparison test and is indicated by asterisks (*p < 0.05, **p < 0.01, ****p < 0.0001).Number of independent images analyzed: nempty(RNAi) = 25, nubc-9(RNAi) = 34, nnpl-4(RNAi) = 38. Scale bars, 10 µm in (A) and (B) and 5 µm in (H).

Fig 3: Cooperation between USP7/MATH-33 and VCP/CDC-48 in DNA replication(A) Analysis of EdU incorporation by immunofluorescence and high-throughput microscopy in HCT116 cells treated with DMSO, 5 µM NMS873 (VCPi), 50 µM P22077 (USP7i), or a combination of both for the indicated time.(B) RPE cells were transfected with a non-specific small interfering RNA (siRNA) (Con, black) or siRNA against FAF1 (FAF1, orange), and the levels of EdU incorporation were measured by high-throughput microscopy 48 h after transfection. Cells were treated with DMSO (Control) or 10 µM P22077 (USP7i) for the indicated times, and we show EdU positive cells. The experiment was repeated 3 times, and one representative result is shown.(C) Schematic representation of measurement of DNA replication checkpoint-mediated cell-cycle delay in C. elegans early embryos.(D) Graph shows synthetic effect of combined ubxn-3 and math-33 inactivation (orange and red bars) compared to the WT control (gray bar). Circles indicate individual data points, bars show mean values, and error bars show standard error of the mean. Asterisks indicate statistical significance in one-way ANOVA Sidak’s multiple-comparison test (**p < 0.01, ***p < 0.001).