Journal: Cancers

Article Title: Acetylated DNMT1 Downregulation and Related Regulatory Factors Influence Metastatic Melanoma Patients Survival

doi: 10.3390/cancers13184691

Figure Lengend Snippet: Ac-DNMT1 and USP7 protein levels positive correlate in metastatic melanoma. ( A ) Representative IHC images of USP7 in the TMA melanoma cohort. Scale bars = 20 μm. ( B , C ) Correlation between USP7 and ac-DNMT1 protein levels in stage III ( B ) or stage IV ( C ) metastatic FFPE tissues. ( D ) Comparison of USP7 H-scores in stage IV patients with high or low ac-DNMT1 protein levels. ( E ) Comparison of USP7 H-scores in normal organ tissues, stage III, and stage IV metastasis from the TMA melanoma cohort. ( F , G ) Correlation between USP7 and TIP60 protein levels in stage III ( F ) or stage IV ( G ) metastatic FFPE tissues. The best-fit line (straight line) and the 95% CI (dotted line) are shown in grey. Data represent the mean ± SD. ns : not significant, * p < 0.05, *** p < 0.001, and **** p < 0.0001.

Article Snippet: IHC staining was performed as previously described [ , ], using anti-human DNMT1 rabbit polyclonal antibody (Ab, 1:100 dilution, Cat# NB100-264, Novus Biologicals, Centennial, CO, USA), anti-human ac-DNMT1 rabbit polyclonal Ab (1:100 dilution, Cat# A5595, ABclonal, Woburn, MA, USA), anti-human Ki-67 mouse monoclonal Ab (1:100 dilution, Cat# M7240, DAKO, Santa Clara, CA, USA), anti-human TIP60 rabbit polyclonal Ab (1:100 dilution, Cat# 10827-1-AP, Proteintech, Rosemont, IL, USA), and anti-human USP7 rabbit monoclonal Ab (1:100 dilution, Cat# A700-072, Bethyl, Montgomery, TX, USA) for the TMA slide.

Techniques: Comparison

Journal: Cancers

Article Title: Acetylated DNMT1 Downregulation and Related Regulatory Factors Influence Metastatic Melanoma Patients Survival

doi: 10.3390/cancers13184691

Figure Lengend Snippet: Low ac-DNMT1 protein level is a predictive factor for poor MSS. ( A ) MSS curves for stage IV metastasis from the TMA cohort. Patients were divided according to the mean IHC H-scores of ac-DNMT1. ( B ) MSS curves for stage IV metastasis from the TMA cohort. Patients were divided according to the mean IHC H-scores of ac-DNMT1 together with DNMT1 ( B ), TIP60 ( C ), USP7 ( D ), and Ki-67 ( E ) into high and low groups, respectively.

Article Snippet: IHC staining was performed as previously described [ , ], using anti-human DNMT1 rabbit polyclonal antibody (Ab, 1:100 dilution, Cat# NB100-264, Novus Biologicals, Centennial, CO, USA), anti-human ac-DNMT1 rabbit polyclonal Ab (1:100 dilution, Cat# A5595, ABclonal, Woburn, MA, USA), anti-human Ki-67 mouse monoclonal Ab (1:100 dilution, Cat# M7240, DAKO, Santa Clara, CA, USA), anti-human TIP60 rabbit polyclonal Ab (1:100 dilution, Cat# 10827-1-AP, Proteintech, Rosemont, IL, USA), and anti-human USP7 rabbit monoclonal Ab (1:100 dilution, Cat# A700-072, Bethyl, Montgomery, TX, USA) for the TMA slide.

Techniques:

Journal: Cancers

Article Title: Acetylated DNMT1 Downregulation and Related Regulatory Factors Influence Metastatic Melanoma Patients Survival

doi: 10.3390/cancers13184691

Figure Lengend Snippet: Multivariate analyses including USP7 protein levels for 4-year MSS 1 in stage IV melanoma patients.

Article Snippet: IHC staining was performed as previously described [ , ], using anti-human DNMT1 rabbit polyclonal antibody (Ab, 1:100 dilution, Cat# NB100-264, Novus Biologicals, Centennial, CO, USA), anti-human ac-DNMT1 rabbit polyclonal Ab (1:100 dilution, Cat# A5595, ABclonal, Woburn, MA, USA), anti-human Ki-67 mouse monoclonal Ab (1:100 dilution, Cat# M7240, DAKO, Santa Clara, CA, USA), anti-human TIP60 rabbit polyclonal Ab (1:100 dilution, Cat# 10827-1-AP, Proteintech, Rosemont, IL, USA), and anti-human USP7 rabbit monoclonal Ab (1:100 dilution, Cat# A700-072, Bethyl, Montgomery, TX, USA) for the TMA slide.

Techniques:

Journal: EMBO Reports

Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

doi: 10.1038/s44319-024-00300-9

Figure Lengend Snippet: ( A , B ) In vitro deubiquitination assay using recombinant WDR48-WDR20-USP12 (WT or C48S) complex, UCHL5 or USP7, and ubiquitinated Itgb1 enriched from USP12/46-dKO ESCRT-KD fibroblast cell lysate followed by WB ( A ) for ubiquitin with quantification ( B ) of Itgb1 ubiquitination levels. The intensity of the Ub signals was normalized to the intensity of the IP-ed Itgb1 signals. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test comparing the BSA group with USP12 WT , USP12 C48S , UCHL5, or USP7 group in ESCRT-KD condition ( P = 0.0194, 0.2707, 0.9997, and 0.9844, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. BSA served as a negative control. ( C ) Coomassie-blue staining of recombinant proteins used in the in vitro deubiquitination assay. MS was used to determine the identity of the protein bands indicated in the DUB complexes. Band *1, WDR48; band *2, WDR20; band *3, a cleaved WDR20; band *4, USP12. ( D ) Representative structured illumination microscopy (SIM) images of Itgb1 and Ub in USP12/46-dKO fibroblasts stably expressing EGFP-USP12 treated with or without ESCRT-KD siRNAs. Boxes indicate magnified cell regions displayed in the Zoom panel. White/pink arrowheads show the Itgb1-labeled FAs, and white arrows show the direction of line profiles. Scale bar, 10 µm. ( E – G ) Superplots showing the PCC between Ub and EGFP-USP12 ( E ), Ub and Itgb1 ( F ), and Itgb1 and EGFP-USP12 ( G ) in USP12/46-dKO fibroblasts stably expressing EGFP-USP12 and treated with or without ESCRT-KD siRNAs. Statistical analysis was carried out by two-sided Welch’s t -test. The P values in ( E – G ) are 0.0287, 0.0011, and 0.0048, respectively. * P < 0.05; ** P < 0.01. Data were shown as Mean ± SD, n = 3 independent experiments, in total 42 cells per condition. ( H ) Representative SIM images of Itgb1 and EEA1 in USP12/46-dKO fibroblasts stably expressing EGFP-USP12 treated with or without ESCRT-KD siRNAs. Boxes indicate magnified cell regions displayed in the Zoom panel. White/pink arrowheads show the Itgb1-labeled FAs, and white arrows show the direction of line profiles. Scale bar, 10 µm. ( I – K ) Superplots showing the PCC between EEA1 and EGFP-USP12 ( I ), EEA1 and Itgb1 ( J ), and Itgb1 and EGFP-USP12 ( K ) in USP12/46-dKO fibroblasts stably expressing EGFP-USP12 and treated with or without ESCRT-KD siRNAs. Statistical analysis was carried out by two-sided Welch’s t -test. The P values in ( I – K ) are 0.0178, 0.0040, and 0.0208, respectively. * P < 0.05; ** P < 0.01. Data were shown as Mean ± SD, n = 3 independent experiments, in total 42 cells per condition. .

Article Snippet: Recombinant USP7 , Novus Biologicals , E-519-025.

Techniques: In Vitro, Recombinant, Ubiquitin Proteomics, Comparison, Negative Control, Staining, Microscopy, Stable Transfection, Expressing, Labeling

Journal: EMBO Reports

Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

doi: 10.1038/s44319-024-00300-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Recombinant USP7 , Novus Biologicals , E-519-025.

Techniques: Recombinant, Expressing, Sequencing, Real-time Polymerase Chain Reaction, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, BIA-KA, Western Blot, Software, Microscopy

Journal: Nature Communications

Article Title: PARylation prevents the proteasomal degradation of topoisomerase I DNA-protein crosslinks and induces their deubiquitylation

doi: 10.1038/s41467-021-25252-9

Figure Lengend Snippet: 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.

Article Snippet: Human recombinant USP7 was purchased from R&D Systems (cat # E-519).

Techniques: Tandem Mass Spectroscopy, Transfection, Expressing, Plasmid Preparation, Control, In Vitro, Recombinant, Ubiquitin Proteomics, Incubation, Western Blot, FLAG-tag, Modification, Quantitation Assay, Generated, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: PARylation prevents the proteasomal degradation of topoisomerase I DNA-protein crosslinks and induces their deubiquitylation

doi: 10.1038/s41467-021-25252-9

Figure Lengend Snippet: a PARP1 and TOP1 form cellular protein complexes (present study and refs. , ). b TOP1-DPC trapped by CPT is rapidly modified with PAR by PARP1 and with ubiquitin (by RNF4 and potentially other E3 ligases, not shown). The PARylation recruits TDP1, PARG, and USP7 to the TOP1-DPC. c TOP1-DPC PARylation is readily and rapidly reversed by PARG, enabling the 26S proteasome to target the ubiquitylated TOP1-DPC for degradation. d TDP1 hydrolyzes the TOP1 peptide to expose the DNA ends for repair. e In the presence of PARGi, TOP1-DPC dePARylation is blocked and the persistent PAR polymers on TOP1-DPC obstruct the proteasome hence stabilize TOP1-DPC. f The stabilization of TOP1-DPC triggers USP7 to deubiquitylate the DPC to recycle the ubiquitin molecules.

Article Snippet: Human recombinant USP7 was purchased from R&D Systems (cat # E-519).

Techniques: Modification, Ubiquitin Proteomics