Journal: Open biology

Article Title: RNF4 and USP7 cooperate in ubiquitin-regulated steps of DNA replication.

doi: 10.1098/rsob.230068

Figure Lengend Snippet: Figure 3. Bortezomib causes elevated cell apoptosis in TKO cells. (a) Representative dose–response curve to bortezomib measured by CellTiter-Glo cell viability assay. Cell survival is normalized to the untreated control. The mean survival with SD of each concentration is shown. The asymmetrical (five-parameter) logistic dose–response model is used to fit the curve. (b) Bortezomib IC50 values. Each data point is an IC50 value from an experiment. Bars and error bars indicate the mean with SD. (c) Cell apoptosis after 48 h of bortezomib measured by Annexin V-PI staining. Each data point is a biological replicate and the mean with SD is shown. (d) Cartoon schematics of constructs used to complement TKO cells. EGFP, enhanced green fluorescent protein; SIM, SUMO-interacting motif; RING, really interesting new gene; TRAF, tumour necrosis factor receptor-associated factor; CD, catalytic domain; UBL, ubiquitin-like. (e) Western blot analyses of whole cell extracts of TKO cells expressing either EGFP-RNF4 or RNF4-EGFP. (f ) Western blot analyses of whole cell extracts of TKO cells expressing WT USP7. (g) Western blot analyses of SUMOylated chromatin-bound proteins of TKO cells expressing either EGFP-RNF4 or RNF4-EGFP. Cells were treated with 10 µM of MG132 and 1 mM of HU for 4 h before collecting. (h) Cell apoptosis after 48 h of bortezomib measured by Annexin V-PI staining in TKO and complemented cells. (b,c,h) Each data point represents the average value from independent plates in an experiment. Bars and error bars indicate the mean and SD across multiple biological experiments. Significance was measured using an ordinary one-way ANOVA with Tukey’s multiple comparisons test; n.s., not significant; **p ≤0.01; ***p ≤0.001.

Article Snippet: To complement RPE-1 USP7−/– cell lines, we transfected the parental cells with pcDNA3.1-N-Myc_WT USP7 (Addgene no. 131242) [96] via Lipofectamine 3000.

Techniques: Viability Assay, Control, Concentration Assay, Staining, Construct, Ubiquitin Proteomics, Western Blot, Expressing

Journal: Open biology

Article Title: RNF4 and USP7 cooperate in ubiquitin-regulated steps of DNA replication.

doi: 10.1098/rsob.230068

Figure Lengend Snippet: Figure 6. RNF4 and USP7 cooperate in ubiquitin-regulated steps of DNA replication. TP53−/– cells have defective G1/S checkpoint, leading to unrestrained G1/S transition even in the presence of DNA damage. With a functional intra-S checkpoint response, cells are capable of pausing and repairing the lesions to prevent premature mitotic entry if cells accumulate too much damage. RNF4, a SUMO-targeted E3 ubiquitin ligase, facilitates the turnover of SUMOylated and ubiquitinated proteins accumulated upon replication stress. USP7, on the other hand, maintains the SUMO and ubiquitin balance at the replication forks by deubiquitinating substrate proteins. These two seemingly opposite actions, however, both sustain the nuclear ubiquitin pool and are particularly critical when proteasome activity is inhibited, where RNF4 and USP7 play a major and a minor role, respectively. Depletion of the nuclear ubiquitin pool suppresses the ATR-mediated checkpoint response upon proteasome inhibition. TKO cells exhibit significantly increased fork asymmetry and reduced DNA synthesis when the proteasome is inhibited. A compromised ATR-mediated checkpoint upon proteasome inhibition permits premature mitotic entry in TKO cells, leading to high levels of anaphase abnormalities and presumably programmed cell death.

Article Snippet: To complement RPE-1 USP7−/– cell lines, we transfected the parental cells with pcDNA3.1-N-Myc_WT USP7 (Addgene no. 131242) [96] via Lipofectamine 3000.

Techniques: Ubiquitin Proteomics, Functional Assay, IF-cells, Activity Assay, Inhibition, DNA Synthesis

Journal: Cell Death & Disease

Article Title: SPOP and HAUSP bidirectionally regulate LZTS2 ubiquitination to modulate the Wnt pathway

doi: 10.1038/s41419-025-08351-z

Figure Lengend Snippet: A HAUSP increased Myc-LZTS2 protein in a dose-dependent manner in 293 T cells. B Knockdown of HAUSP decreased endogenous LZTS2, YAP and GLI3 protein levels in HCT-116 cells. C 293 T cells were treated with CHX (20 μg/ml) for the indicated times. Of note, knockdown of HAUSP promoted the degradation of endogenous LZTS2. D The deubiquitinase-deficiency mutant HAUSP-CA failed to increase Myc-LZTS2 protein. E HAUSP enhanced endogenous LZTS2 protein in a dose-dependent manner in HT-29 cells, which was abolished by treatment with 10 μM of HAUSP inhibitor P22077 for 24 h. F Treatment with three HAUSP inhibitors (P5091, USP7-IN-1, or P22077 ) at the indicated concentrations for 24 h apparently downregulated endogenous LZTS2 protein in HCT-116 cells. G Immunoblots of immunoprecipitates (top two panels) or whole cell extracts (bottom three panels) from 293 T cells transfected with the indicated plasmids. HAUSP suppressed K48- and K63-linked polyubiquitination on LZTS2. Above all, Actin acts as a loading control.

Article Snippet: For HAUSP inhibitors treatment, cells were treated by P22077 (#HY-13865, MedChemExpress), USP7-IN (#HY-16709, MedChemExpress) or P5091 (#SML0070, Sigma) at indicated concentrations for 24 h.

Techniques: Knockdown, Mutagenesis, Western Blot, Transfection, Control