vcp inhibitors  (MedChemExpress)

Journal: Nature Communications

Article Title: A small molecule VDAC ligand inhibits ERAD and induces selective cancer cell death via disruption of calcium homeostasis

doi: 10.1038/s41467-025-67816-z

Figure Lengend Snippet: a ER dislocation can be visualized using NHK-drGFP reporter. Live cell imaging of HeLa cells expressing SP-S11-NHK-HA (ERAD client) and S1-10 (cytoplasmic complementary fragment of GFP). Bortezomib is added to block proteasomal degradation and measure ER dislocation (green). CellMask Deep Red Plasma Membrane Stain and Hoechst 33342 (blue) were used to stain the plasma membrane and nucleus, respectively. NMS-873, a p97 inhibitor, blocks ER dislocation. Imaging was performed using an Opera Phenix High Content Screening System. Scale bar = 100 microns. Images shown are representative of at least three independent experiments. b NMS-873 inhibits ER dislocation and NHK-drGFP fluorescence. A 16-dose titration of NMS-873 was included as a control on six 1536-well assay plates. Fluorescence was measured using an Acumen Laser Scanning Imaging Cytometer, and the IC 50 is indicated. c High-throughput screening of 265 assay plates. NHK-drGP fluorescence was measured for bortezomib (black circles, neutral control, mean ± SD, n = 32 wells per plate) and bortezomib plus NMS-873 (red squares, inhibitory control, mean ± SD, n = 16 wells per plate). d HTS screening funnel describing triage of 96,047 compound screen. Created in BioRender. Henderson, Mark. ( https://BioRender.com/iwzn2wx ) e Six compounds from HTS prioritized for follow-up testing. The compounds were tested at 11-concentrations using two ERAD substrates, NHK and CD3δ. f Effect of compounds on CFTR biogenesis was examined. BHK cells stably expressing HA-∆F508 CFTR were treated with DMSO or different doses of compounds for 24 h followed by processing for immunoblotting. Two distinct CFTR forms were detected: the ER-resident core-glycosylated B form and the fully glycosylated mature C form, representing different stages of CFTR processing and trafficking. Data shown are representative of three independent experiments.

Article Snippet: To monitor assay performance, each plate included a 16 pt titration of the p97 inhibitor, NMS-873 (HY-15713; MedChemExpress), as a reference control.

Techniques: Live Cell Imaging, Expressing, Blocking Assay, Clinical Proteomics, Membrane, Staining, Imaging, High Content Screening, Fluorescence, Titration, Control, Cytometry, High Throughput Screening Assay, Stable Transfection, Western Blot

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: DAF-16/FOXO and HLH-30/TFEB comprise a cooperative regulatory axis controlling tubular lysosome induction in C. elegans

doi: 10.1038/s41467-025-64832-x

Figure Lengend Snippet: a Endogenously tagged SPIN-1::mCherry in starved L1 worms with and without dSVIP overexpression. Scale bar, 5 µm. b Quantification of lysosomal junctions per object for the genotypes indicated. WT ( N = 20 worms), WT + dSVIP , daf-16(mu86) , daf-16(mu86) + dSVIP , hlh-30(tm1978) and hlh-30(tm1978) + dSVIP ( N = 30 worms per genotype) , daf-16(mu86); hlh-30(tm1978) and daf-16(mu86); hlh-30(tm1978) + dSVIP ( N = 20 worms per genotype). Mean ± s.e.m. One-way ANOVA with Šídák’s multiple comparisons test. (ns = not significant, ** p < 0.01, **** p < 0.0001) ( c ) Endogenously tagged SPIN-1::mCherry in fed day 1 adult worms with and without dSVIP overexpression. Scale bar, 5 µm. d Quantification of lysosomal junctions per object for the genotypes indicated. WT ( N = 28 worms), WT + dSVIP ( N = 20 worms), daf-16(mu86) ( N = 28 worms), daf-16(mu86) + dSVIP ( N = 28 worms), hlh-30(tm1978) ( N = 29 worms), hlh-30(tm1978) + dSVIP ( N = 25 worms) , daf-16(mu86); hlh-30(tm1978) ( N = 28 worms), daf-16(mu86); hlh-30(tm1978) + dSVIP ( N = 29 worms). Mean ± s.e.m. One-way ANOVA with Šídák’s multiple comparisons test. (ns = not significant, **** p < 0.0001). e Endogenously tagged SPIN-1::mCherry in fed WT ( N2 ) and daf-16(mu86) worms with gut dSVIP OE at day 1 of adulthood that were fed control DMSO or CB5083 VCP inhibitor beginning at L4 larval stage. Scale bar, 5 µm. f Quantification of lysosomal junctions per object for the genotypes and conditions indicated. WT ( N2 ) + DMSO ( N = 19 worms), daf-16(mu86); gut dSVIP OE + DMSO ( N = 18 worms), WT ( N2 ) + CB5083 ( N = 15 worms), daf-16(mu86); gut dSVIP OE + CB5083 ( N = 15 worms). Mean ± s.e.m. One-way ANOVA with Šídák’s multiple comparisons test. (**** p < 0.0001). g Endogenously tagged SPIN-1::mCherry in day 7 WT ( N2 ) animals that were fed DMSO or CB5083 VCP inhibitor beginning at day 5 of adulthood. Scale bar, 5 µm. h Quantification of lysosomal junctions per object. ( N = 20 control worms and N = 16 fed CB5083 worms). Mean ± s.e.m. Unpaired two-tailed Student’s t -test. (ns = not significant). i Endogenously tagged SPIN-1::mCherry in day 1 eat-2(ad1116) adults that were fed DMSO or CB5083 VCP inhibitor beginning at L4 larval stage. Scale bar, 5 µm. j Quantification of lysosomal junctions per object. eat-2(ad1116) +DMSO ( N = 20 worms), eat-2(ad1116) +CB5083 ( N = 16 worms). Mean ± s.e.m. Unpaired two-tailed Student’s t -test. (ns = not significant). For all experiments, data were pooled from two independent experiments.

Article Snippet: A 10 μM stock solution of the VCP inhibitor CB5083 (MedChem Express, Cat. # HY-12861/CS-5405) was prepared in DMSO and diluted to a final working concentration of 1 μM in M9 buffer.

Techniques: Over Expression, Control, 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 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