sirna targeting usp10 and plk1  (Shanghai GenePharma)

Journal: The Journal of Biological Chemistry

Article Title: USP10 inhibits the degradation of α-synuclein, a pathogenic factor associated with Parkinson's disease, by inhibiting chaperone-mediated autophagy

doi: 10.1016/j.jbc.2025.110292

Figure Lengend Snippet: Reduction of USP10 decreases endogenous α-synuclein (α-syn) levels in cells. A – C , SH-SY5Y cells were transfected with three different USP10-siRNAs (siUSP10) or nontargeting siRNA (siNT) using Lipofectamine RNAiMAX. Whole-cell lysates were analyzed by Western blotting using anti-α-syn, anti-USP10, and anti-β-actin antibodies. The ratio of the α-syn band to the β-actin band was measured by densitometry, and the mean and SD from three experiments are presented in B and C , respectively. The significance of the differences was assessed by a one-way ANOVA followed by Dunnett's multiple comparisons test. ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. D – F , SH-SY5Y cells were grown on coverslips, transfected with three different siUSP10 or siNT, and stained with anti-α-syn ( green ) and anti-USP10 ( red ) antibodies, whereas nuclei were stained with Hoechst 33258 ( blue ). The fluorescence intensity of α-syn ( E ) and USP10 ( F ) was measured by fluorescence microscopy, and the ratio of intensity for each knockdown sample compared with the control was calculated from 100 cells and presented as mean ± SD. The significance of the differences was assessed by a one-way ANOVA followed by Dunnett's multiple comparisons test. ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. Scale bars represent 20 μm. USP10, ubiquitin-specific protease 10.

Article Snippet: siRNAs specific to human USP10 (Oligo IDs: HSS113446, HSS113447, and HSS113448) and negative control siRNA (catalog no.: 12935-100) were purchased from Thermo Fisher Scientific.

Techniques: Transfection, Western Blot, Staining, Fluorescence, Microscopy, Knockdown, Control, Ubiquitin Proteomics

Journal: Cell Death & Disease

Article Title: USP10 promotes the progression and attenuates gemcitabine chemotherapy sensitivity via stabilizing PLK1 in PDAC

doi: 10.1038/s41419-025-07757-z

Figure Lengend Snippet: A Flow chart of the USP10 substrate screening process. B A Venn diagram showing that PLK1 is the only substrate of USP10. C The interaction between USP10 and PLK1 was confirmed through molecular docking analysis. D Exogenous CO-IP assay of USP10 and PLK1 in HEK293 cells. E , F Endogenous interaction of USP10 and PLK1 detected via Co-IP assay in PDAC cells. G The direct interaction between USP10 and PLK1 was validated using a GST pull-down assay. H HEK293 cells were transfected with the respective plasmids, and full length and fragments of USP10 were used to pull down full-length PLK1. I HEK293 cells were transfected with the respective plasmids, and full length and fragments of PLK1 were used to pull down full-length USP10.

Article Snippet: The siRNA targeting USP10 and PLK1 were purchased from GenePharma Co., Ltd, and the sequences were provided in supplementary Table .

Techniques: Co-Immunoprecipitation Assay, Pull Down Assay, Transfection

Journal: Cell Death & Disease

Article Title: USP10 promotes the progression and attenuates gemcitabine chemotherapy sensitivity via stabilizing PLK1 in PDAC

doi: 10.1038/s41419-025-07757-z

Figure Lengend Snippet: A The change of USP10 and PLK1 protein after transfection with USP10 siRNAs in PDAC cells. B The protein level of USP10 and PLK1 were detected via western blot after transfection with HA-USP10 plasmids. C PANC-1 cells were transfected with si-NC, siUSP10-1, and siUSP10-2 and treated with 20 μM MG132 for 24 h, and the protein level of USP10 and PLK1 were detected through western blot. D PANC-1 cells were transfected with different siRNAs and treated with 10 μg/ml CHX for 0 h, 4 h, 8 h, and 12 h. The change of USP10 and PLK1 proteins were detected. The PANC1 E and MIAPaCa-2 F cells were treated with protein synthesis inhibitor CHX and autophagy inhibitor CQ, and the protein level of PLK1 was detected. G Myc-USP10 and other plasmids were transfected into HEK293 cells and treated with 20 μM MG132 for 6 h. The ubiquitination level of PLK1 was detected via IP assay. H Myc-USP10 (C424A) and other plasmids were transfected into HEK293 cells and treated with 20 μM MG132 for 6 h. The ubiquitination level of PLK1 was detected via IP assay. I The ubiquitinated Flag-PLK1 was purified from HEK293, and GST-USP10 was purified from E. coli BL21 (DE3). Next, the two proteins were incubated in the deubiquitination buffer at 37 °C for 2 h. The ubiquitination level of Flag-PLK1 was detected through western blot.

Article Snippet: The siRNA targeting USP10 and PLK1 were purchased from GenePharma Co., Ltd, and the sequences were provided in supplementary Table .

Techniques: Transfection, Western Blot, Ubiquitin Proteomics, Purification, Incubation

Journal: Cell Death & Disease

Article Title: USP10 promotes the progression and attenuates gemcitabine chemotherapy sensitivity via stabilizing PLK1 in PDAC

doi: 10.1038/s41419-025-07757-z

Figure Lengend Snippet: A , B Confirmation of PLK1 knockdown efficiency. C , D CCK-8 assay was used to assess the impact of PLK1 knockdown on the proliferation. E , F EdU assay was employed to assess the impact of PLK1 knockdown on the proliferation. G , H Influence of PLK1 knockdown on the colony formation ability. I , J The change in migration ability after PLK1 knockdown confirmed through wound healing assay. K , L Influence of PLK1 knockdown on the migration and invasion ability, detected using Transwell assay. Data are presented as mean ± sd. from three biologically independent samples. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The siRNA targeting USP10 and PLK1 were purchased from GenePharma Co., Ltd, and the sequences were provided in supplementary Table .

Techniques: Knockdown, CCK-8 Assay, EdU Assay, Migration, Wound Healing Assay, Transwell Assay

Journal: Cell Death & Disease

Article Title: USP10 promotes the progression and attenuates gemcitabine chemotherapy sensitivity via stabilizing PLK1 in PDAC

doi: 10.1038/s41419-025-07757-z

Figure Lengend Snippet: A , B The si-NC, siUSP10-1, siUSP10-2, and the overexpression plasmid of PLK1 were transfected into PDAC cells as required. The EBSS medium was used to activate autophagy. The autophagy-related proteins were detected. C , D PDAC cells were infected with the lentivirus Mcherry-EGFP-LC3B. Next, si-USP10 and the overexpression plasmid of PLK1 were transfected into PDAC cells as required, and the cells were treated with EBSS medium for 8 h before being observed under a confocal microscope. E si-USP10 and the overexpression plasmid of PLK1 were transfected into PDAC cells according to the requirement, and the cells were treated with EBSS medium for 8 h before being observed under a transmission electron microscope. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The siRNA targeting USP10 and PLK1 were purchased from GenePharma Co., Ltd, and the sequences were provided in supplementary Table .

Techniques: Over Expression, Plasmid Preparation, Transfection, Infection, Microscopy, Transmission Assay

Journal: Molecular Cancer

Article Title: CircHAS2 activates CCNE2 to promote cell proliferation and sensitizes the response of colorectal cancer to anlotinib

doi: 10.1186/s12943-024-01971-7

Figure Lengend Snippet: Interaction and co-localization of circHAS2 with USP10. A The enrichment of circHAS2-MS2 complex formation was detected with MS2-CP-Flag in SW620 cells. B The Silver staining of circHAS2-associated proteins by the biotin-labeled probe. C The RNA pull-down confirmed the interaction between circHAS2 and USP10. The relative expression of circHAS2 was determined using the RIP assay with AGO2 and IgG antibodies in SW620 cells. D The RIP assays were conducted under the specified treatment conditions using anti-USP10 and anti-IgG antibodies in SW620 cells. E Representative images of the FISH assay in SW620 cells revealed the colocalization of circHAS2 and USP10 in the cytoplasm with the target probe labeled with Cy3 and nuclei stained with DAPI (scale bar: 20 μm). F The colocalization of circHAS2 and USP10 in CRC tissues and paired normal tissues of microarrays were verified by FISH (scale bar: 400 μm; scale bar: 100 μm). G The interaction profile between circHAS2 fragments and USP10 was predicted by the catRAPID database. H The interaction between circHAS2 full-length and truncations with USP10 was validated using RIP assays in HEK-293T cells. I The heat map illustrated the interaction between circHAS2 and the N-terminal region (1-100 amino acids) of USP10 by the catRAPID database. Statistical significance in two-group experiments was assessed using a two-sided Student’s t-test, and multiple-group comparisons were conducted through one-way analysis of variance. Data are represented as mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance

Article Snippet: USP10 siRNA was synthesized by Sangon (Sangon Biotech, China).

Techniques: Silver Staining, Labeling, Expressing, Staining

Journal: Molecular Cancer

Article Title: CircHAS2 activates CCNE2 to promote cell proliferation and sensitizes the response of colorectal cancer to anlotinib

doi: 10.1186/s12943-024-01971-7

Figure Lengend Snippet: CircHAS2 interaction with USP10 to enhance p53 Ubiquitination. A Schematic diagram of USP10 full-length and truncations. B The RIP assay was performed in SW620 cells using anti-Flag antibodies to assess the expression levels of circHAS2 following transfection with USP10 full-length and truncations. C The RNA pull-down assays were performed using biotin-labeled circHAS2 probes to capture interacting proteins and detect the expression of USP10 full-length and truncations by representative Western blotting images in SW620 cells. D The direct interaction between USP10 and p53 was analyzed by Co-IP assays with USP10 and p53 antibodies in SW620 cells. E The Co-IP assays were conducted under the specified treatment conditions using USP10 and p53 antibodies in SW620 cells. F After indicated treatments were treated with various durations of cycloheximide (0.1 mg/ml) in SW620 cells, and the expression of p53 was detected by Western blotting. GAPDH was employed as an internal control. G CircHAS2 modulated the levels of p53 ubiquitination through the interaction with USP10. SW620 cells were transfected with the indicated constructs and subjected to MG132 (50 mM) for 4 h before harvest to evaluate the ubiquitination levels of p53. H Representative images of the FISH assay revealed the localization of p53 after indicated treatments with the target probe labeled with Cy3 and nuclei stained with DAPI in SW620 cells (scale bar: 20 μm). I Quantification of cells with different p53 subcellular localization after indicated treatments in SW620 cells. Nuc, Nucleus only; Cyto + Nuc, both cytoplasm and nucleus. J SW620 cells were transfected with the indicated constructs and subjected to MG132 treatment to evaluate the ubiquitination levels of cytoplasmic or nuclear p53. K Relative p53 and p21 expressions were verified after indicated treatments in SW620 cells. L Representative Western blotting images with indicated treatments in SW620 cells for Flag, p53, and p21 proteins. GAPDH was used as an internal control. Statistical significance in two-group experiments was assessed using a two-sided Student’s t-test. Data are represented as mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance

Article Snippet: USP10 siRNA was synthesized by Sangon (Sangon Biotech, China).

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Labeling, Western Blot, Co-Immunoprecipitation Assay, Control, Construct, Staining

Journal: Molecular Cancer

Article Title: CircHAS2 activates CCNE2 to promote cell proliferation and sensitizes the response of colorectal cancer to anlotinib

doi: 10.1186/s12943-024-01971-7

Figure Lengend Snippet: CircHAS2 activating bidirectional downstream pathways to promote CRC progression. A The viabilities of SW620 cells following indicated treatments were detected by CCK-8 assays. B Quantitative results and representative images of cell proliferation were conducted by colony formation assay in SW620 cells following indicated treatments. C Quantitative results and representative images of cell proliferation were evaluated by EdU incorporation in SW620 cells following indicated treatments (scale bar: 50 μm). D The percentage of cells in the G1, S, and G2 phases of the entire cell population was determined by flow cytometry following indicated treatments in SW620 cells. E Representative Western blotting images with indicated treatments for p53, p21, CDK2, and CCNE2 proteins in SW620 cells. GAPDH was used as an internal control. F IHC scores of ISH staining (miR-1244) and IHC staining (USP10, p53, p21, CCNE2, and CDK2) assay of tumors of indicated treatments. Cell viability was determined by CCK-8 assay, employing two-way analysis of variance. Statistical differences in clone formation, EdU experiments, and IHC scores were assessed using two-sided Student’s t-test. Data are represented as mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance

Article Snippet: USP10 siRNA was synthesized by Sangon (Sangon Biotech, China).

Techniques: CCK-8 Assay, Colony Assay, Flow Cytometry, Western Blot, Control, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Transcription factor ATMIN facilitates chemoresistance in nasopharyngeal carcinoma

doi: 10.1038/s41419-024-06496-x

Figure Lengend Snippet: A Silver staining of FLAG-immunoprecipitated proteins separated from HONE1 cells overexpressing FLAG-ATMIN. Black lines indicated the proteins of interest. B Co-IP with anti-FLAG and anti-HA antibodies in HEK293T cells overexpressing FLAG-ATMIN and HA-USP10. C Co-IP with anti-ATMIN and anti-USP10 antibodies in HONE1 and SUNE1 cells. D Immunofluorescence staining revealed the cellular location of USP10 (red) and ATMIN (green) in HONE1 and SUNE1 cells. E Western blot analysis of ATMIN expression with USP10 overexpression or silencing in HONE1 and SUNE1 cells. F The effect of CHX treatment (left) and greyscale analysis of the results (right) in HONE1 and SUNE1 cells transfected with si-USP10#2 or si-NC. G The effect of MG132 (left) and CQ (right) treatment in HONE1 and SUNE1 cells transfected with indicated siRNA. H HEK293T cells (left) co-transfected with FLAG-ATMIN, HA-ubiquitin (Ub) and MYC-USP10 or the vector plasmids were subjected to Co-IP and immunoblotted with the indicated antibodies. HONE1 (middle) and SUNE1 (right) cells co-transfected with FLAG-ATMIN, HA-ubiquitin (Ub) and si-USP10#2 or si-NC were subjected to Co-IP and immunoblotted with the indicated antibodies. Data in ( F ) are presented as mean ± SD, P values were calculated using Student’s t test, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The unprocessed images of the blots are shown in Supplementary Fig. .

Article Snippet: Meanwhile, the pCMV-kana-Ub (WT)-HA plasmid was acquired from Vigene Bioscience (China), and the USP10 siRNA (si-USP10) and ATMIN siRNA (si-ATMIN) were purchased from RiboBio (China).

Techniques: Silver Staining, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Western Blot, Expressing, Over Expression, Transfection, Ubiquitin Proteomics, Plasmid Preparation