usp10 inhibitor spautin 1  (MedChemExpress)

Journal: Acta Pharmaceutica Sinica. B

Article Title: USP10-mediated deubiquitination and activation of KRAS mutants promotes colorectal cancer via a novel USP10/KRAS positive feedback circuit

doi: 10.1016/j.apsb.2025.11.015

Figure Lengend Snippet: Identification of USP10 as an essential regulator of KRAS mutants. (A) The schematic diagram of DUB inhibitors (DUBi) library screening and identification Spautin-1 selectively inhibiting KRAS -mutant CRC cell growth. HT-29 cells were infected with the lentivirus that stably over-expressed HT-29-KRAS WT/G12C/G12D/G12V ( HT-29-KRAS WT/Mut ) and screened by using puromycin, then these cells were seeded with 1000 cells/well in 96-well plates, and then were treated with 10 μmol/L DUB inhibitor for 72 h, followed by SRB assays. (B) The underlying proteins interacting with oncogenic KRAS were identified by LC–MS/MS. HEK-293T cells were transfected with KRAS G12V -Flag and vector for 24 h, then cells were lysed with RIPA lysis buffer and incubated with anti-Flag agarose beads. The immunoprecipitated KRAS-Flag was captured using SDS-PAGE and the gel was subjected to trypsin digestion for LC–MS/MS analysis. (C) The Venn diagram illustrate the potential targets that are involved in oncogenic KRAS-induced cell proliferation and interact with KRAS mutants. (D) USP10 predominantly interacts with mutant KRAS in vivo. HEK-293T cells were co-transfected with indicated expression plasmids and the cell lysates were subjected to IP assays. (E) USP10 interacts with KRAS mutants at endogenous levels. Cell lysates of SW480 and SW620 were incubated with the affinity gel conjugated with KRAS antibody. Proteins retained on the affinity gel were subjected to IB analysis.

Article Snippet: USP10 inhibitor Spautin-1 (#HY-12990) and other DUB inhibitors were purchased from MedChemExpress (NJ, USA).

Techniques: Library Screening, Mutagenesis, Infection, Stable Transfection, Liquid Chromatography with Mass Spectroscopy, Transfection, Plasmid Preparation, Lysis, Incubation, Immunoprecipitation, SDS Page, In Vivo, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: USP10-mediated deubiquitination and activation of KRAS mutants promotes colorectal cancer via a novel USP10/KRAS positive feedback circuit

doi: 10.1016/j.apsb.2025.11.015

Figure Lengend Snippet: USP10 positively regulates KRAS mutants' activity. (A) Depletion of USP10 posed little effect on KRAS protein levels. SW480 and SW620 cells were infected with the lentivirus encoding the indicated shRNA, and the cell lysates were subjected to IB analysis. (B) Overexpression of USP10 posed little effect on KRAS protein levels. SW480 and SW620 cells were transfected with USP10-WT or USP10-CA, and the cell lysates were subjected to IB analysis. (C) Depletion of USP10 posed little effect on KRAS mRNA levels. SW480 and SW620 cells were infected with the lentivirus encoding the indicated shRNA, and cell lysates were subjected to real-time PCR assays (Mean ± SD, n = 3). (D) Depletion of USP10 remarkably sustained MAPK signaling. SW480 and SW620 cells were infected with indicated sh RNA s, and the cell lysates were subjected to IB analysis. (E) USP10 inhibitor Spautin-1 significantly suppressed MAPK signaling. SW480 and SW620 cells were treated with 25 μmol/L Spautin-1 for the indicated time, and the cell lysates were subjected to IB analysis. (F) Overexpression of USP10 significantly promoted MAPK signaling in an enzymatic activity-dependent manner. SW480 and SW620 cells were transfected with the indicated plasmid, and the cell lysates were subjected to IB analysis. n.s., P > 0.05.

Article Snippet: USP10 inhibitor Spautin-1 (#HY-12990) and other DUB inhibitors were purchased from MedChemExpress (NJ, USA).

Techniques: Activity Assay, Infection, shRNA, Over Expression, Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Acta Pharmaceutica Sinica. B

Article Title: USP10-mediated deubiquitination and activation of KRAS mutants promotes colorectal cancer via a novel USP10/KRAS positive feedback circuit

doi: 10.1016/j.apsb.2025.11.015

Figure Lengend Snippet: USP10 directly deubiquitinates KRAS in an enzymatic activity-dependent manner. (A, B) Overexpression of USP10-WT, but not USP10-CA, significantly decreases KRAS mutants' ubiquitination levels. Cells transfected with the indicated plasmids were treated with MG132 (10 μmol/L, 8 h) before harvested. Total cell lysates were immunoprecipitated with anti-Flag affinity gels, then the proteins remaining on gels were subjected to IB analysis. (C) Depletion of USP10 dramatically increased KRAS mutants' ubiquitination. HEK-293T cells infected with control/USP10 shRNA were transfected with the indicated plasmids and treated with MG132 (10 μmol/L, 8 h) before harvested. Cell lysates were immunoprecipitated with anti-Flag affinity gels, and the proteins remaining on gels were subjected to IB analysis. (D) USP10 inhibitor Spautin-1 significantly increased KRAS mutants' ubiquitination. HEK-293T cells transfected with the indicated plasmids were treated with MG132 (10 μmol/L, 8 h) and Spautin-1 (50 μmol/L, 10 h) before harvested. Cell lysates were immunoprecipitated with anti-Flag affinity gels, then the protein remaining on gels were immunoblotted with the indicated antibodies. (E) Bacterial-expressed recombinant human USP10 (rhUSP10) effectively removed the polyubiquitination of KRAS mutants in vitro . HEK-293T cells were transfected with indicated expression plasmids and cell lysates were incubated with anti-Flag affinity gel for overnight. Then, proteins retained on anti-Flag affinity gel were incubated with rhUSP10 in 37 °C for 2 h, followed by IB assays. (F) Overexpression of USP10 efficiently removes the K6-, K11-, K27- and K29-linked polyubiquitin chains of KRAS mutants. Cells transfected with the indicated plasmids were treated with MG132 (10 μmol/L, 8 h) before harvested. Total cell lysates were immunoprecipitated with anti-Flag affinity gels, then the proteins remaining on gels were immunoblotted with the indicated antibodies.

Article Snippet: USP10 inhibitor Spautin-1 (#HY-12990) and other DUB inhibitors were purchased from MedChemExpress (NJ, USA).

Techniques: Activity Assay, Over Expression, Ubiquitin Proteomics, Transfection, Immunoprecipitation, Infection, Control, shRNA, Recombinant, In Vitro, Expressing, Incubation

Journal: Acta Pharmaceutica Sinica. B

Article Title: USP10-mediated deubiquitination and activation of KRAS mutants promotes colorectal cancer via a novel USP10/KRAS positive feedback circuit

doi: 10.1016/j.apsb.2025.11.015

Figure Lengend Snippet: USP10 knockdown significantly suppresses KRAS -mutant CRC growth in vitro and in vivo . (A, B) Depletion of USP10 suppressed the proliferation of KRAS -mutant CRC cells in vitro . SW620, SW480 and HCT-116 cells were infected the lentivirus that encoding control/USP10 shRNA, followed by cell proliferation assays and colony formation assays (mean ± SD; n = 3). (C–E) Depletion of USP10 significantly arrested the growth of KRAS -mutant CRC xenografts in vivo . (C) The bearing-tumor mice and tumor images. (D) The relative tumor volume of the indicated groups. (E) The tumor weight of indicated groups (mean ± SEM; n = 10/group). (F) USP10 knockdown remarkably inhibited intratumor MAPK signaling. Immunoblot analysis of related protein levels in SW480 xenografts and SW620 xenografts. (G) The schematic diagram of PDX models. (H, I) Depletion of USP10 significantly inhibited the growth of KRAS -mutant CRC PDX in vivo (mean ± SEM; n = 6/group). (H) The relative tumor volume of indicated groups. (I) The tumor weight of indicated groups. (J) USP10 knockdown remarkably restrained intratumor MAPK signaling. Immunoblot analysis of related protein levels in PDXs. (K) USP10 levels in tumor tissues are significantly higher than those of in normal tissues in CRC patients. (L) USP10 is significantly overexpressed in tumor tissue compared to normal tissue in CRC patients. Representative section of H&E staining and USP10 staining in CRC patient tissues. T: tumor tissues; N: para-carcinoma tissues; scale bars: 250 μm. (M) USP10 level is inversely correlated with the overall survival of CRC patients. The correlation analysis between USP10 expression and overall survival of colorectal cancer patients in the Kaplan–Meier Plotter database (split by the best cutoff). ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: USP10 inhibitor Spautin-1 (#HY-12990) and other DUB inhibitors were purchased from MedChemExpress (NJ, USA).

Techniques: Knockdown, Mutagenesis, In Vitro, In Vivo, Infection, Control, shRNA, Western Blot, Staining, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: USP10-mediated deubiquitination and activation of KRAS mutants promotes colorectal cancer via a novel USP10/KRAS positive feedback circuit

doi: 10.1016/j.apsb.2025.11.015

Figure Lengend Snippet: KRAS mutants upregulates USP10 levels and stability by promoting the phosphorylation of USP10 at Tyr42 and Ser337. (A) KRAS -mutant CRC patients displayed higher USP10 levels compared to KRAS wild-type patients. Immunohistochemistry (IHC) staining of intratumor USP10 in KRAS mutants/wild-type CRC patients (mean ± SD, n = 6). (B) Overexpression of KRAS mutants significantly upregulated USP10 protein levels. HT-29 cells were transfected with the indicated plasmids and cell lysates were subjected to IB assays. (C) Overexpression of KRAS mutants, not KRAS wild-type, significantly promoted USP10 phosphorylation. HEK-293T cells transfected with indicated plasmids were immunoprecipitated with anti-Flag agarose beads and the protein remaining on anti-Flag agarose beads were subjected to IB assays. (D) MAPK inhibitor PD98059 reversed the upregulation of USP10 phosphorylation induced by KRAS mutants. HEK-293T cells transfected with indicated plasmids were treated with DMSO, PD98059 (10 μmol/L) for 24 h before harvested, then cell lysates were immunoprecipitated with anti-Flag agarose beads and immunoblotted with the indicated antibody. (E) T42 and S337 are the main phosphorylation sites of USP10 regulated by KRAS mutants. HEK-293T cells transfected with indicated plasmids were immunoprecipitated with anti-Flag agarose beads and the protein remaining on anti-Flag agarose beads was subjected to IB assays. TA&SA: T42A&S337A. (F) KRAS mutants significantly increased the protein levels and stability of USP10-WT, but not USP10-T42A&S337A. HT-29 cells expressing USP10-WT/TA&SA were infected with lentivirus encoding Vector or KRAS G12V -Myc and treated with 20 μg/mL CHX for indicated time and the cell lysates were immunoblotted with the indicated antibody. TA&SA: T42A&S337A. ∗∗ P < 0.01.

Article Snippet: USP10 inhibitor Spautin-1 (#HY-12990) and other DUB inhibitors were purchased from MedChemExpress (NJ, USA).

Techniques: Phospho-proteomics, Mutagenesis, Immunohistochemistry, Over Expression, Transfection, Immunoprecipitation, Expressing, Infection, Plasmid Preparation

Journal: Acta Pharmaceutica Sinica. B

Article Title: USP10-mediated deubiquitination and activation of KRAS mutants promotes colorectal cancer via a novel USP10/KRAS positive feedback circuit

doi: 10.1016/j.apsb.2025.11.015

Figure Lengend Snippet: USP10 inhibition preferentially inhibits the growth of KRAS -mutant CRC in vitro and in vivo . (A, B) Depletion of USP10 selectively inhibited KRAS -mutant CRC proliferation. HT-29 cells stably expressing KRAS WT/G12C/G12D/G12V were infected with the lentivirus encoding control/USP10 shRNA and then these cells were subjected to cell proliferation assays (A) and colony formation assays (B). (C–E) Depletion of USP10 selectively suppressed KRAS -mutant CRC xenografts in vivo (mean ± SEM, n = 10/group). (C) The tumor images of indicated groups. (D) The relative tumor volume of indicated groups. (E) The tumor weight of indicated groups. ∗ P < 0.05; ∗∗∗ P < 0.001.

Article Snippet: USP10 inhibitor Spautin-1 (#HY-12990) and other DUB inhibitors were purchased from MedChemExpress (NJ, USA).

Techniques: Inhibition, Mutagenesis, In Vitro, In Vivo, Stable Transfection, Expressing, Infection, Control, shRNA

Journal: Acta Pharmaceutica Sinica. B

Article Title: USP10-mediated deubiquitination and activation of KRAS mutants promotes colorectal cancer via a novel USP10/KRAS positive feedback circuit

doi: 10.1016/j.apsb.2025.11.015

Figure Lengend Snippet: USP10 is elevated in KRAS -mutant CRC tissues. (A, B) IHC assays were performed to detect the expression of USP10 and KRAS in KRAS -mutant ( n = 36) and wild-type ( n = 36) CRC tissues. (C) KRAS -mutant CRC tissues displayed higher USP10 levels than KRAS wild-type. IHC score was evaluated by multiplication of positive staining proportions (1 score, <25%; 2 score, 25%–50%; 3 score, 50%–75%; 4 score, 75%–100%) and protein expression intensity (1 score, weak staining; 2 score, moderate staining; 3 score, high staining). –, negative expression (1–3 score); +, low expression (4–6 score); ++, medium expression (7–9 score); +++, high expression (10–12 score). (D) Statistical analysis of USP10 expression in KRAS wild-type/mutant CRC specimens. (E) The schematic diagram of USP10/KRAS positive feedback circuit promoting the proliferation of KRAS -mutant CRC. ∗∗∗ P < 0.001.

Article Snippet: USP10 inhibitor Spautin-1 (#HY-12990) and other DUB inhibitors were purchased from MedChemExpress (NJ, USA).

Techniques: Mutagenesis, Expressing, Staining

Journal: International Journal of Biological Sciences

Article Title: Co-targeting MRPS7-23 synergistically enhances cisplatin efficacy to suppress nasopharyngeal carcinoma growth and metastasis

doi: 10.7150/ijbs.115523

Figure Lengend Snippet: USP10 depletion reduces expression of MRPS7 and MRPS23 and suppresses xenograft tumor growth. (A-C) The protein of USP10 co-immunoprecipitated with MRPS7 and MRPS23 was verified by mass spectrometry. (D) The co-localization of MRPS7 (green), MRPS23 (red) and USP10 (purple) detected by immunofluorescence assay in NPC cells. The scale bar is 10 μm. (E) The binding interface between MRPS7 (yellow) and USP10 (purple) was predicted using computational molecular docking. (F) The binding interface between MRPS23 (yellow) and USP10 (purple) was predicted using computational molecular docking. (G) Direct interaction between USP10 and MRPS7 measured by surface plasmon resonance. (H) Direct interaction between USP10 and MRPS23 measured by surface plasmon resonance. (I) OS curves of low and high expressed of USP10. (J) Bioinformatic analysis of TCGA data revealed differential expression of USP10 in HNSC tumors compared to normal tissues. (K) Western blot analysis of MRPS7, MRPS23 and USP10 protein levels in NPC cells with or without knockdown of USP10 after the treatment with MG132. (L) Following co-transfection with HA-Ub-K48O and either an empty vector or Flag-MRPS7/23 plasmids, NPC cells were treated with MG132 and lysates were immunoprecipitated under denaturing conditions with the specified antibodies. (M) Western blot analysis was conducted to assess MRPS7, MRPS23, and β-catenin expression in USP10-knockdown NPC cells, with or without concomitant overexpression of MRPS7 and MRPS23. (N) The photo of excised Cne2 tumors. (O) Tumor volumetric measurements were evaluated at predetermined intervals. (P) The measurement of tumor weight in excised Cne2 tumors. (Q-R) Ki67 staining and expression of tumor tissue. * p < 0.05 and *** p < 0.001.

Article Snippet: For western blot analysis, After SDS-PAGE (10%) and PVDF transfer (Millipore), membranes were blocked (5% milk, 1 h RT) then incubated with primary antibodies (4°C, overnight): MRPS7 (1:1000, Abcam, UK), MRPS23 (1:1000, Proteintech, USA), β-catenin (1:1000, Proteintech, USA), Slug (1:1000, Proteintech, USA), Vimentin (1:1000, Proteintech, USA), E-cadherin (1:1000, Proteintech, USA), USP10 (1:1000, Proteintech, USA), SOX2 (1:1000, Proteintech, USA), Oct4 (1:1000, Abcam, UK), and Nanog (1:1000, Abcam, UK).

Techniques: Expressing, Immunoprecipitation, Mass Spectrometry, Immunofluorescence, Binding Assay, SPR Assay, Quantitative Proteomics, Western Blot, Knockdown, Cotransfection, Plasmid Preparation, Over Expression, Staining

Journal: International Journal of Biological Sciences

Article Title: Co-targeting MRPS7-23 synergistically enhances cisplatin efficacy to suppress nasopharyngeal carcinoma growth and metastasis

doi: 10.7150/ijbs.115523

Figure Lengend Snippet: Therapeutic targeting of USP10 by spautin-1 potentiates cisplatin efficacy to suppress tumor growth and metastasis in NPC. (A-B) Cne2 and C666 cells were incubated with Spautin-1, cisplatin, or combination therapy at graded concentrations for 48 h, with viability determined by CCK-8 assay. (C-D) Synergistic effects of Spautin-1/Cisplatin combination in Cne2 and C666 cells were quantitatively analyzed using CalcuSyn software. (E-F) Dose-dependent combination indexes (CIs) and fraction affected (Fa) values for Spautin-1/Cisplatin treatment in both cell lines are presented. (G-I) The Spautin-1/Cisplatin combination significantly suppressed the migratory capacity of NPC cells. (J) Images of resected Cne2 xenograft tumors. (K) Tumor growth kinetics were monitored at specified time points. (L) Tumor weight of excised Cne2 tumors. (M) The mice were weighted on the indicated days. (N) Ki67, slug, E-cadherin and SOX2 staining and expression of tumor tissue. (O) The relative expression of Ki67, slug, E-cadherin and SOX2 in tumor tissue. (P) Schematic diagram of spautin-1 and cisplatin treatment on NPC lung metastasis. (Q-R) H&E-stained lung tissue of lung metastasis model, with subsequent quantification of microscopically detectable metastatic foci. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: For western blot analysis, After SDS-PAGE (10%) and PVDF transfer (Millipore), membranes were blocked (5% milk, 1 h RT) then incubated with primary antibodies (4°C, overnight): MRPS7 (1:1000, Abcam, UK), MRPS23 (1:1000, Proteintech, USA), β-catenin (1:1000, Proteintech, USA), Slug (1:1000, Proteintech, USA), Vimentin (1:1000, Proteintech, USA), E-cadherin (1:1000, Proteintech, USA), USP10 (1:1000, Proteintech, USA), SOX2 (1:1000, Proteintech, USA), Oct4 (1:1000, Abcam, UK), and Nanog (1:1000, Abcam, UK).

Techniques: Incubation, CCK-8 Assay, Software, Staining, Expressing

Journal: International Journal of Biological Sciences

Article Title: Co-targeting MRPS7-23 synergistically enhances cisplatin efficacy to suppress nasopharyngeal carcinoma growth and metastasis

doi: 10.7150/ijbs.115523

Figure Lengend Snippet: MRPS7, MRPS23, and USP10 expression predicts poor outcomes and chemoresistance in nasopharyngeal carcinoma. (A-C) Immunohistochemical (IHC) staining showing different intensities of MRPS7, MRPS23, and USP10 expression in NPC tissues. Scale bar, 100 μm. (D-E) Comparative IHC analysis of MRPS7, MRPS23, and USP10 expression in tumor versus adjacent non-tumor tissues (n = 41). Scale bar, 100 μm. (F) IHC images of MRPS7, MRPS23, and USP10 in NPC patient samples. Scale bar, 100 μm. (G) Correlation analysis of MRPS7 with MRPS23, MRPS7 with USP10, and MRPS23 with USP10 in NPC tissues (n = 41). (H-I) Kaplan-Meier survival curves demonstrating that high expression of MRPS7, MRPS23, and USP10 is associated with shorter disease-free survival. (J) MRPS7, MRPS23 and USP10 expression, as evaluated by IHC staining, was associated with the response to cisplatin treatment.

Article Snippet: For western blot analysis, After SDS-PAGE (10%) and PVDF transfer (Millipore), membranes were blocked (5% milk, 1 h RT) then incubated with primary antibodies (4°C, overnight): MRPS7 (1:1000, Abcam, UK), MRPS23 (1:1000, Proteintech, USA), β-catenin (1:1000, Proteintech, USA), Slug (1:1000, Proteintech, USA), Vimentin (1:1000, Proteintech, USA), E-cadherin (1:1000, Proteintech, USA), USP10 (1:1000, Proteintech, USA), SOX2 (1:1000, Proteintech, USA), Oct4 (1:1000, Abcam, UK), and Nanog (1:1000, Abcam, UK).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry