p97 inhibitor cb 5083  (MedChemExpress)

Journal: bioRxiv

Article Title: Minute-scale control of ubiquitin-mediated degradation reveals dynamics of bacterial secreted effector-functions

doi: 10.1101/2025.11.19.688170

Figure Lengend Snippet: (A) Immunofluorescence microscopy confirms Cdu1 depletion dynamics. Cdu1 (green, anti-FLAG), chlamydial inclusion (Hsp60,red), and DNA (DAPI, blue). Scale bar = 10 µm. (B) Expansion microscopy (4× expansion) resolving Cdu1 localization patterns under undegraded and degraded conditions. Scale bar = 10 µm. (C) Cdu1 degradation depends on ubiquitin-proteasome and p97 pathways. Host cells were pretreated with inhibitors for 3 hours (including 1 hour during 5-Ph-IAA treatment) with pan-E1 (TAK-243, 1 µM), proteasome (MG-132, 10 µM; bortezomib, 1 µM), or p97 (CB-5083, 10 µM) inhibitors. Degradation was blocked despite 1-hour 5-Ph-IAA exposure. (D) Quantification of inhibitor effects on Cdu1 degradation. FLAG levels (normalized to OmpA, mean ± SD) from three independent immunoblot replicates (one representative in C). Significance assessed by one-way ANOVA with Tukey Multiple comparisons test (*p < 0.05; n.s., not significant). (E) Immunofluorescence validation of inhibitors. Inclusion-localized Cdu1 signal persists in treated cultures. Scale bar = 10 µm. All experiments were replicated ≥3 times with consistent results.

Article Snippet: Proteasome inhibitors (MG-132 at 10 μM, bortezomib at 1 μM; Cell Signaling), p97 inhibitor CB-5083 (10 μM; MedChemExpress), and ubiquitin-activating enzyme inhibitor TAK-243 (1 μM; TAK-243) were prepared as 1,000× stock solutions in DMSO.

Techniques: Immunofluorescence, Microscopy, Ubiquitin Proteomics, Western Blot, Biomarker Discovery

Journal: Nature Communications

Article Title: USP37 prevents unscheduled replisome unloading through MCM complex deubiquitination

doi: 10.1038/s41467-025-59770-7

Figure Lengend Snippet: a Model displaying the molecular players involved in replication termination. During replication termination, the CMG replicative helicase (CDC45-MCM2-7-GINS) is poly-ubiquitinated with lysine 48 (K48) ubiquitin linkages, and the replisome is disassembled through p97. A deubiquitinase (DUB) could antagonize the ubiquitination-dependent disassembly to prevent premature replisome disassembly. b Workflow for chromatin flow cytometry assays to study replication and bound MCM. RPE1 cells were treated for 24 h with either p97i or a panel of siRNAs to knock down selected DUBs individually (siDUB). Cells were labeled with EdU (thymidine analog) 30 min prior to harvesting, then soluble proteins were pre-extracted to retain only chromatin-bound proteins such as MCM2 (one of the replisome components). Cells were then fixed and stained for EdU (for active DNA synthesis), MCM2 (as a representative subunit for the MCM2-7 complex), and DAPI (for total DNA content) for flow cytometric analysis. c Chromatin flow cytometry for RPE1 cells treated with 20 nM siControl or 1.25 μM of CB-5083 (p97 inhibitor) for 24 h, and pulsed with EdU for 30 min before harvesting. Cells were stained for bound MCM2, and DAPI (for DNA content). In the late S/G2/M gate, control cells are divided into high ( > 10 3 ) versus low ( < 10 3 ) bound MCM. Representative of two biological replicates. d Histograms of the late S/G2/M-MCM DNA -positive cells from (C). e RPE1 cells were treated with siControl or siDUB at 20 nM as indicated. Box and whisker plots for EdU intensity per cell in S phase. Box represents 25 th −75 th percentile with line at median. Cells in each sample were randomly down-sampled to 2400 cells per sample. Data is combined from two independent biological replicates. Relative fold-change of the means of EdU intensity from the two replicates was computed: siControl versus siUSP37, unpaired two tailed t test, p = 0.0115. Source data are provided as a Source Data file. f Bound MCM in late S/G2/M from cells treated as in ( e ). Left: Histograms of normalized counts of the late S/G2/M-MCM DNA -positive cells. Representative of one biological replicate. Right: Relative percentage of high MCM, late S/G2/M-MCM DNA -positive cells computed from at least two independent biological replicates; mean with error bars ± SEM. Unpaired two tailed t test for the means of the three replicates for siControl versus siUSP37, p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: For the experiment described in Figs. b, , cells were treated with 5 μM of the p97 inhibitor CB-5083 (Selleck Chem Cat. #S8101) for the final 4 h prior to harvesting.

Techniques: Ubiquitin Proteomics, Flow Cytometry, Knockdown, Labeling, Staining, DNA Synthesis, Control, Whisker Assay, Two Tailed Test

Journal: Nature Communications

Article Title: USP37 prevents unscheduled replisome unloading through MCM complex deubiquitination

doi: 10.1038/s41467-025-59770-7

Figure Lengend Snippet: a USP37 was depleted using siRNA for 48 h in RPE1 cells stably expressing a 6xHis-FLAG-tagged ubiquitin construct. Ubiquitinated proteins were pulled down using Ni-NTA, revealing that USP37 siRNA increases endogenous MCM7 ubiquitination, as observed by immunoblotting. Representative of two biological replicates. b MCM7 ubiquitination was analyzed as described in ( a ), except that cells were treated with 5 µM of the p97i CB-5083 for the last 4 h before harvesting. Inhibition of p97 strongly increases MCM7 ubiquitination, and this is even more pronounced after USP37 depletion. Representative of three biological replicates. c HeLa cells stably expressing CDC45 GFP were depleted of USP37 before being stabilized prior to S phase using thymidine. After release, cells were treated with DMSO or 5 µM of p97i before CMG complexes were isolated using biotinylated anti-GFP Nanobodies (Nb) conjugated to Strep-tacin resin. Inhibition of p97 combined with depletion of USP37 significantly enhanced ubiquitination of endogenous MCM7. Representative of two biological replicates. d Ubiquitinated MCM7 isolated from HEK-293T cells was mixed with 100 nM of recombinant USP37 WT or a catalytically inactive mutant (C350S). The in vitro deubiquitination assay shows that USP37 WT, but not C350S, deubiquitinates Ub-MCM7. Representative of > three biological replicates. e FLAG-tagged USP37 was ectopically expressed for 48 hours and subsequently purified from HEK-293T cells by FLAG immunoprecipitation. USP37 immunoprecipitates were mixed with 1 µM of K11, K48, or K63 tetra-ubiquitin chains, revealing that USP37 cleaves Tetra- and Tri-Ub more efficiently than Di-Ub. Representative of two biological replicates.

Article Snippet: For the experiment described in Figs. b, , cells were treated with 5 μM of the p97 inhibitor CB-5083 (Selleck Chem Cat. #S8101) for the final 4 h prior to harvesting.

Techniques: Stable Transfection, Expressing, Ubiquitin Proteomics, Construct, Western Blot, Inhibition, Isolation, Recombinant, Mutagenesis, In Vitro, Purification, Immunoprecipitation

Journal: Nature Communications

Article Title: USP37 prevents unscheduled replisome unloading through MCM complex deubiquitination

doi: 10.1038/s41467-025-59770-7

Figure Lengend Snippet: a Model displaying the molecular players involved in replication termination. During replication termination, the CMG replicative helicase (CDC45-MCM2-7-GINS) is poly-ubiquitinated with lysine 48 (K48) ubiquitin linkages, and the replisome is disassembled through p97. A deubiquitinase (DUB) could antagonize the ubiquitination-dependent disassembly to prevent premature replisome disassembly. b Workflow for chromatin flow cytometry assays to study replication and bound MCM. RPE1 cells were treated for 24 h with either p97i or a panel of siRNAs to knock down selected DUBs individually (siDUB). Cells were labeled with EdU (thymidine analog) 30 min prior to harvesting, then soluble proteins were pre-extracted to retain only chromatin-bound proteins such as MCM2 (one of the replisome components). Cells were then fixed and stained for EdU (for active DNA synthesis), MCM2 (as a representative subunit for the MCM2-7 complex), and DAPI (for total DNA content) for flow cytometric analysis. c Chromatin flow cytometry for RPE1 cells treated with 20 nM siControl or 1.25 μM of CB-5083 (p97 inhibitor) for 24 h, and pulsed with EdU for 30 min before harvesting. Cells were stained for bound MCM2, and DAPI (for DNA content). In the late S/G2/M gate, control cells are divided into high ( > 10 3 ) versus low ( < 10 3 ) bound MCM. Representative of two biological replicates. d Histograms of the late S/G2/M-MCM DNA -positive cells from (C). e RPE1 cells were treated with siControl or siDUB at 20 nM as indicated. Box and whisker plots for EdU intensity per cell in S phase. Box represents 25 th −75 th percentile with line at median. Cells in each sample were randomly down-sampled to 2400 cells per sample. Data is combined from two independent biological replicates. Relative fold-change of the means of EdU intensity from the two replicates was computed: siControl versus siUSP37, unpaired two tailed t test, p = 0.0115. Source data are provided as a Source Data file. f Bound MCM in late S/G2/M from cells treated as in ( e ). Left: Histograms of normalized counts of the late S/G2/M-MCM DNA -positive cells. Representative of one biological replicate. Right: Relative percentage of high MCM, late S/G2/M-MCM DNA -positive cells computed from at least two independent biological replicates; mean with error bars ± SEM. Unpaired two tailed t test for the means of the three replicates for siControl versus siUSP37, p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: The following chemicals/inhibitors were used in this study: Doxycycline (dox) (CalBiochem, cat. # 32485) was used at 2.5 or 5 ng/mL for the rescue experiments, and 100 or 25 ng/mL for the cyclin E1 or c-MYC overproduction experiments; ATR inhibitor (ATRi) AZD6738 (Selleck, cat. #S7693) was used at 5 μM; p97 inhibitor (Selleck, cat. #S8101) was used at 1.25 μM for the flow cytometry experiments and 5 μM for biochemical experiments; hydroxyurea (HU) drug (Selleck, cat. #S1896) was used at 150 μM for the HU block and release experiments.

Techniques: Ubiquitin Proteomics, Flow Cytometry, Knockdown, Labeling, Staining, DNA Synthesis, Control, Whisker Assay, Two Tailed Test

Journal: Nature Communications

Article Title: USP37 prevents unscheduled replisome unloading through MCM complex deubiquitination

doi: 10.1038/s41467-025-59770-7

Figure Lengend Snippet: a USP37 was depleted using siRNA for 48 h in RPE1 cells stably expressing a 6xHis-FLAG-tagged ubiquitin construct. Ubiquitinated proteins were pulled down using Ni-NTA, revealing that USP37 siRNA increases endogenous MCM7 ubiquitination, as observed by immunoblotting. Representative of two biological replicates. b MCM7 ubiquitination was analyzed as described in ( a ), except that cells were treated with 5 µM of the p97i CB-5083 for the last 4 h before harvesting. Inhibition of p97 strongly increases MCM7 ubiquitination, and this is even more pronounced after USP37 depletion. Representative of three biological replicates. c HeLa cells stably expressing CDC45 GFP were depleted of USP37 before being stabilized prior to S phase using thymidine. After release, cells were treated with DMSO or 5 µM of p97i before CMG complexes were isolated using biotinylated anti-GFP Nanobodies (Nb) conjugated to Strep-tacin resin. Inhibition of p97 combined with depletion of USP37 significantly enhanced ubiquitination of endogenous MCM7. Representative of two biological replicates. d Ubiquitinated MCM7 isolated from HEK-293T cells was mixed with 100 nM of recombinant USP37 WT or a catalytically inactive mutant (C350S). The in vitro deubiquitination assay shows that USP37 WT, but not C350S, deubiquitinates Ub-MCM7. Representative of > three biological replicates. e FLAG-tagged USP37 was ectopically expressed for 48 hours and subsequently purified from HEK-293T cells by FLAG immunoprecipitation. USP37 immunoprecipitates were mixed with 1 µM of K11, K48, or K63 tetra-ubiquitin chains, revealing that USP37 cleaves Tetra- and Tri-Ub more efficiently than Di-Ub. Representative of two biological replicates.

Article Snippet: The following chemicals/inhibitors were used in this study: Doxycycline (dox) (CalBiochem, cat. # 32485) was used at 2.5 or 5 ng/mL for the rescue experiments, and 100 or 25 ng/mL for the cyclin E1 or c-MYC overproduction experiments; ATR inhibitor (ATRi) AZD6738 (Selleck, cat. #S7693) was used at 5 μM; p97 inhibitor (Selleck, cat. #S8101) was used at 1.25 μM for the flow cytometry experiments and 5 μM for biochemical experiments; hydroxyurea (HU) drug (Selleck, cat. #S1896) was used at 150 μM for the HU block and release experiments.

Techniques: Stable Transfection, Expressing, Ubiquitin Proteomics, Construct, Western Blot, Inhibition, Isolation, Recombinant, Mutagenesis, In Vitro, Purification, Immunoprecipitation

Journal: eLife

Article Title: Early recovery of proteasome activity in cells pulse-treated with proteasome inhibitors is independent of DDI2

doi: 10.7554/eLife.91678

Figure Lengend Snippet: ( a ) Expression of DDI2 in the CRISPR-generated clones of HAP1 cells used in this work was analyzed by western blot. ( b ) The experimental setup used in this study. Cells were pulse treated with bortezomib (Btz) or carfilzomib (Cfz) for 1 hr, then cultured in drug-free media for times indicated and analyzed as described. ( c ) The viability of wt- and DDI2 KO clones of HAP1 cells was measured using CellTiter-Glo, and the inhibition of β5 sites was measured with the Proteasome-Glo assay at times indicated; n=2–5. ( d ) Knockout of DDI2 inhibits the Nrf1 processing. Western blots of Btz-treated HAP1 cells. The sample in the first lane is wt cells treated with VCP/p97 inhibitor CB-5083 immediately after removal of Btz. VCP inhibitors blocks Nrf1 processing ( ; ; ). ( e ) MDA-MB-231 and SUM149 cells were analyzed by western blot 72 hr after transfection with DDI2 siRNAs ( f ) Theβ5 activity in siRNA-transfected SUM149 and MDA-MB-231 was measured using Suc-LLVY-AMC immediately and 18 hr after treatment with 100 nM Btz; n=3. ( g ) β5 activity was measured in HCT-116 cells with the Proteasome-Glo assay immediately and 18 hr after treatment with PIs; n=2. Figure 1—source data 1. PDF file containing and original full-size western blot membranes (anti-DDI2, anti-GAPDH) with molecular weight markers. Figure 1—source data 2. Excel file containing data for . Figure 1—source data 3. PDF file containing and original full-size western blot membranes (anti-Nrf1, anti-DDI2, anti-β-actin) with molecular weight markers. Figure 1—source data 4. PDF file containing and full-size western blot membranes (anti-DDI2, anti-β-actin). Additional lanes demonstrate that the knockdown of DDI2 is maintained throughout the experiment. Figure 1—source data 5. Excel file containing data and statistical analysis for . Figure 1—source data 6. Excel file containing data for .

Article Snippet: Chemical compound, drug , CB-5083 , Cayman Chemicals , CAS# 1542705-92-9, Cat# 19311 , p97 inhibitor,.

Techniques: Expressing, CRISPR, Generated, Clone Assay, Western Blot, Cell Culture, Inhibition, Glo Assay, Knock-Out, Transfection, Activity Assay, Molecular Weight, Knockdown

Journal: eLife

Article Title: Early recovery of proteasome activity in cells pulse-treated with proteasome inhibitors is independent of DDI2

doi: 10.7554/eLife.91678

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , CB-5083 , Cayman Chemicals , CAS# 1542705-92-9, Cat# 19311 , p97 inhibitor,.

Techniques: Generated, CRISPR, Mutagenesis, Transfection, Construct, Sequencing, Bradford Assay, Isolation, Reverse Transcription, SYBR Green Assay, Viability Assay, Software