usp10 expression  (Santa Cruz Biotechnology)

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: MedComm

Article Title: Obestatin Treatment Counteracts Muscle Wasting by Reactivation of Autophagy in Duchenne Muscular Dystrophy

doi: 10.1002/mco2.70563

Figure Lengend Snippet: Autoubiquitination of NEDD4‐L facilitate activation of autophagy through the VPS34 stabilization in human DMD myotubes. (A) Representative images from VPS34 and NEDD4‐L immunostaining of DMD myotubes treated with obestatin (10 nM) or insulin (1.72 µM). The changes in MFI of VPS34 and NEDD4‐L are shown. Pearson's coefficient ( r ) indicates the correlation of intensity values of green and red pixels in dual‐channel images. Data were expressed as mean ± SEM ( n = 5 per group; * p < 0.05). (B) Analysis of VPS34 ubiquitination in DMD cells transfected with HA‐NEDD4‐L or HA‐NEDD4L DD, and treated with obestatin (10 nM, 3 h) in the presence of MG132. VPS34 was immunoprecipitated by using anti‐VPS34 antibody followed by immunoblot analysis with antiubiquitin, antiubiquitin K48 linkage, antiubiquitin K63 linkage, UPS10 or VPS34 antibody. Data were expressed as mean ± SEM ( n = 3 per group; *,# p < 0.05). (C) Coimmunoprecipitation and immunoblot analysis of extracts of DMD and KM155C25 cells treated with obestatin (10 nM, 3 h) in the presence of MG132 using anti‐NEDD4‐L antibody and immunoblotted with antiubiquitin K48 linkage, antiubiquitin K63 linkage, anti‐pY, anti‐USP10, anti‐USP13, anti‐VPS34, or anti‐NEDD4‐L antibody. Data were expressed as mean ± SEM ( n = 3 per group; *,# p < 0.05). (D) Coimmunoprecipitation analysis of DMD cells transfected with control or NEDD4‐L siRNAs and treated with obestatin (10 nM, 3 h) by using anti‐VPS34 antibody and immunoblotted with USP10, VPS34, or NEDD4‐L antibody. Data were expressed as mean ± SEM ( n = 3 per group; *,# p < 0.05). (E) Immunoprecipitation analysis of the VPS34 ubiquitination in DMD cells transfected with control or USP10 siRNAs and treated with obestatin (10 nM, 3 h) in the presence of MG132. Analysis of ubiquitin K48 and K63 linkage was developed by immunoblot. Data were expressed as mean ± SEM ( n = 3 per group; *,# p < 0.05).

Article Snippet: To knockdown NEDD4, AMPKα or USP10 expression, siRNA specifically targeting human NEDD4‐L (sc‐75894; Santa Cruz Biotechnology, CA, USA), AMPKα (sc‐45312; Santa Cruz Biotechnology), or USP10 (sc‐76811; Santa Cruz Biotechnology) was used. siRNA duplexes targeting preproghrelin or c‐Src were selected from ON‐TARGET plus SMARTpool siRNA from Thermo Fisher Scientific (Dharmacon, CO, USA). shRNA‐mediated gene silencing was used for ULK1 knockdown (sc‐44182‐SH; Santa Cruz Biotechnology) in DMD cells.

Techniques: Activation Assay, Immunostaining, Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, Control

Journal: MedComm

Article Title: Obestatin Treatment Counteracts Muscle Wasting by Reactivation of Autophagy in Duchenne Muscular Dystrophy

doi: 10.1002/mco2.70563

Figure Lengend Snippet: USP10 is required for deubiquitination of VPS34 and AMPKα in human DMD myotubes. (A) Immunoblot analysis of pAMPK(T172), AMPK, pULK1(S318), ULK1, NEDD4‐L, VPS34, Beclin1, LC3, and p62 in DMD myotubes transfected with sicontrol or siUSP10 and treated with obestatin (10 nM, 3 h). Data were expressed as the mean ± SEM ( n = 3 per group; *,# p < 0.05). (B) Immunoblot analysis of Beclin1, VPS34, pAMPK(T172), and AMPK in DMD cells transfected with HA‐USP10 or HA‐USP10 CA and treated with obestatin (10 nM, 3 h). Data were expressed as the mean ± SEM obtained from intensity scans ( n = 3; *,# p < 0.05). (C) Immunoprecipitation analysis of the AMPKα ubiquitination in DMD cells after obestatin (10 nM, 20 min) treatment and in the presence of MG132. Analysis of ubiquitin K48 and K63 linkage was developed by immunoblot. Data were expressed as mean ± SEM ( n = 3 per group; * p < 0.05). (D) Coimmunoprecipitation analysis of DMD cells treated with obestatin (10 nM, 20 min) by using anti‐USP10 antibody and immunoblotted with anti‐pAMPK(T172), anti‐AMPKα, anti‐NEDD4‐L, or anti‐USP10 antibody. Data were expressed as mean ± SEM ( n = 3 per group; * p < 0.05). (E) Coimmunoprecipitation analysis of DMD cells treated with obestatin (10 nM, 20 min) by using anti‐AMPKα antibody and immunoblotted with anti‐USP10, anti‐NEDD4‐L, anti‐pAMPK(T172), or anti‐AMPKα antibody. Data were expressed as mean ± SEM ( n = 3 per group; *,# p < 0.05). In panels (A)–(E), immunoblots are representative of the mean value.

Article Snippet: To knockdown NEDD4, AMPKα or USP10 expression, siRNA specifically targeting human NEDD4‐L (sc‐75894; Santa Cruz Biotechnology, CA, USA), AMPKα (sc‐45312; Santa Cruz Biotechnology), or USP10 (sc‐76811; Santa Cruz Biotechnology) was used. siRNA duplexes targeting preproghrelin or c‐Src were selected from ON‐TARGET plus SMARTpool siRNA from Thermo Fisher Scientific (Dharmacon, CO, USA). shRNA‐mediated gene silencing was used for ULK1 knockdown (sc‐44182‐SH; Santa Cruz Biotechnology) in DMD cells.

Techniques: Western Blot, Transfection, Immunoprecipitation, Ubiquitin Proteomics

Journal: MedComm

Article Title: Obestatin Treatment Counteracts Muscle Wasting by Reactivation of Autophagy in Duchenne Muscular Dystrophy

doi: 10.1002/mco2.70563

Figure Lengend Snippet: Tyrosine switch on NEDD4‐L facilitates activation of autophagy through the USP10 recruitment and the VPS34 stabilization in human DMD myotubes. (A) Immunoblot analysis of pc‐Src(Y416), c‐Src, pAMPK(T172), or AMPK in DMD myotubes transfected with sicontrol or si‐c‐Src and treated with obestatin (10 nM, 3 h). Data were expressed as the mean ± SEM ( n = 3 per group; * p < 0.05). (B) Coimmunoprecipitation analysis of DMD cells treated with obestatin (10 nM, 20 min) by using anti‐NEDD4‐L antibody and immunoblotted with ubiquitin, pY, pc‐Src(Y416), USP10, VPS34, or NEDD4‐L antibody. Data were expressed as mean ± SEM ( n = 3 per group; * p < 0.05). In panels (A) and (B), immunoblots are representative of the mean value. (C) Proposed model by which NEDD4‐L regulates autophagy via obestatin signaling under dystrophic conditions. Tyrosine switch on NEDD4‐L activates autoubiquitination that serves as a scaffold to recruit the deubiquitinase enzyme USP10 to form a deubiquitination complex, which stabilizes VPS34 to promote autophagy through the removal of the ubiquitin chains on VPS34 and activation of Beclin1 complex. In parallel, NEDD4‐L favors AMPK exposure to CaMKKß and consequent activation of ULK1. Under DMD conditions, AMPK does not inhibit mTORC1, but sustains ULK1/Beclin1 activity and autophagy. In nonpathological conditions, lack of action of non‐RTK on NEDD4‐L favors interaction between c‐Src and NEDD4‐L, thus impairing the VPS34 stabilization and AMPK activation.

Article Snippet: To knockdown NEDD4, AMPKα or USP10 expression, siRNA specifically targeting human NEDD4‐L (sc‐75894; Santa Cruz Biotechnology, CA, USA), AMPKα (sc‐45312; Santa Cruz Biotechnology), or USP10 (sc‐76811; Santa Cruz Biotechnology) was used. siRNA duplexes targeting preproghrelin or c‐Src were selected from ON‐TARGET plus SMARTpool siRNA from Thermo Fisher Scientific (Dharmacon, CO, USA). shRNA‐mediated gene silencing was used for ULK1 knockdown (sc‐44182‐SH; Santa Cruz Biotechnology) in DMD cells.

Techniques: Activation Assay, Western Blot, Transfection, Ubiquitin Proteomics, Activity Assay

Journal: MedComm

Article Title: Obestatin Treatment Counteracts Muscle Wasting by Reactivation of Autophagy in Duchenne Muscular Dystrophy

doi: 10.1002/mco2.70563

Figure Lengend Snippet: Autoubiquitination of NEDD4‐L facilitate activation of autophagy through the VPS34 stabilization in human DMD myotubes. (A) Representative images from VPS34 and NEDD4‐L immunostaining of DMD myotubes treated with obestatin (10 nM) or insulin (1.72 µM). The changes in MFI of VPS34 and NEDD4‐L are shown. Pearson's coefficient ( r ) indicates the correlation of intensity values of green and red pixels in dual‐channel images. Data were expressed as mean ± SEM ( n = 5 per group; * p < 0.05). (B) Analysis of VPS34 ubiquitination in DMD cells transfected with HA‐NEDD4‐L or HA‐NEDD4L DD, and treated with obestatin (10 nM, 3 h) in the presence of MG132. VPS34 was immunoprecipitated by using anti‐VPS34 antibody followed by immunoblot analysis with antiubiquitin, antiubiquitin K48 linkage, antiubiquitin K63 linkage, UPS10 or VPS34 antibody. Data were expressed as mean ± SEM ( n = 3 per group; *,# p < 0.05). (C) Coimmunoprecipitation and immunoblot analysis of extracts of DMD and KM155C25 cells treated with obestatin (10 nM, 3 h) in the presence of MG132 using anti‐NEDD4‐L antibody and immunoblotted with antiubiquitin K48 linkage, antiubiquitin K63 linkage, anti‐pY, anti‐USP10, anti‐USP13, anti‐VPS34, or anti‐NEDD4‐L antibody. Data were expressed as mean ± SEM ( n = 3 per group; *,# p < 0.05). (D) Coimmunoprecipitation analysis of DMD cells transfected with control or NEDD4‐L siRNAs and treated with obestatin (10 nM, 3 h) by using anti‐VPS34 antibody and immunoblotted with USP10, VPS34, or NEDD4‐L antibody. Data were expressed as mean ± SEM ( n = 3 per group; *,# p < 0.05). (E) Immunoprecipitation analysis of the VPS34 ubiquitination in DMD cells transfected with control or USP10 siRNAs and treated with obestatin (10 nM, 3 h) in the presence of MG132. Analysis of ubiquitin K48 and K63 linkage was developed by immunoblot. Data were expressed as mean ± SEM ( n = 3 per group; *,# p < 0.05).

Article Snippet: To knockdown NEDD4, AMPKα or USP10 expression, siRNA specifically targeting human NEDD4‐L (sc‐75894; Santa Cruz Biotechnology, CA, USA), AMPKα (sc‐45312; Santa Cruz Biotechnology), or USP10 (sc‐76811; Santa Cruz Biotechnology) was used. siRNA duplexes targeting preproghrelin or c‐Src were selected from ON‐TARGET plus SMARTpool siRNA from Thermo Fisher Scientific (Dharmacon, CO, USA). shRNA‐mediated gene silencing was used for ULK1 knockdown (sc‐44182‐SH; Santa Cruz Biotechnology) in DMD cells.

Techniques: Activation Assay, Immunostaining, Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, Control

Journal: MedComm

Article Title: Obestatin Treatment Counteracts Muscle Wasting by Reactivation of Autophagy in Duchenne Muscular Dystrophy

doi: 10.1002/mco2.70563

Figure Lengend Snippet: USP10 is required for deubiquitination of VPS34 and AMPKα in human DMD myotubes. (A) Immunoblot analysis of pAMPK(T172), AMPK, pULK1(S318), ULK1, NEDD4‐L, VPS34, Beclin1, LC3, and p62 in DMD myotubes transfected with sicontrol or siUSP10 and treated with obestatin (10 nM, 3 h). Data were expressed as the mean ± SEM ( n = 3 per group; *,# p < 0.05). (B) Immunoblot analysis of Beclin1, VPS34, pAMPK(T172), and AMPK in DMD cells transfected with HA‐USP10 or HA‐USP10 CA and treated with obestatin (10 nM, 3 h). Data were expressed as the mean ± SEM obtained from intensity scans ( n = 3; *,# p < 0.05). (C) Immunoprecipitation analysis of the AMPKα ubiquitination in DMD cells after obestatin (10 nM, 20 min) treatment and in the presence of MG132. Analysis of ubiquitin K48 and K63 linkage was developed by immunoblot. Data were expressed as mean ± SEM ( n = 3 per group; * p < 0.05). (D) Coimmunoprecipitation analysis of DMD cells treated with obestatin (10 nM, 20 min) by using anti‐USP10 antibody and immunoblotted with anti‐pAMPK(T172), anti‐AMPKα, anti‐NEDD4‐L, or anti‐USP10 antibody. Data were expressed as mean ± SEM ( n = 3 per group; * p < 0.05). (E) Coimmunoprecipitation analysis of DMD cells treated with obestatin (10 nM, 20 min) by using anti‐AMPKα antibody and immunoblotted with anti‐USP10, anti‐NEDD4‐L, anti‐pAMPK(T172), or anti‐AMPKα antibody. Data were expressed as mean ± SEM ( n = 3 per group; *,# p < 0.05). In panels (A)–(E), immunoblots are representative of the mean value.

Article Snippet: To knockdown NEDD4, AMPKα or USP10 expression, siRNA specifically targeting human NEDD4‐L (sc‐75894; Santa Cruz Biotechnology, CA, USA), AMPKα (sc‐45312; Santa Cruz Biotechnology), or USP10 (sc‐76811; Santa Cruz Biotechnology) was used. siRNA duplexes targeting preproghrelin or c‐Src were selected from ON‐TARGET plus SMARTpool siRNA from Thermo Fisher Scientific (Dharmacon, CO, USA). shRNA‐mediated gene silencing was used for ULK1 knockdown (sc‐44182‐SH; Santa Cruz Biotechnology) in DMD cells.

Techniques: Western Blot, Transfection, Immunoprecipitation, Ubiquitin Proteomics

Journal: MedComm

Article Title: Obestatin Treatment Counteracts Muscle Wasting by Reactivation of Autophagy in Duchenne Muscular Dystrophy

doi: 10.1002/mco2.70563

Figure Lengend Snippet: Tyrosine switch on NEDD4‐L facilitates activation of autophagy through the USP10 recruitment and the VPS34 stabilization in human DMD myotubes. (A) Immunoblot analysis of pc‐Src(Y416), c‐Src, pAMPK(T172), or AMPK in DMD myotubes transfected with sicontrol or si‐c‐Src and treated with obestatin (10 nM, 3 h). Data were expressed as the mean ± SEM ( n = 3 per group; * p < 0.05). (B) Coimmunoprecipitation analysis of DMD cells treated with obestatin (10 nM, 20 min) by using anti‐NEDD4‐L antibody and immunoblotted with ubiquitin, pY, pc‐Src(Y416), USP10, VPS34, or NEDD4‐L antibody. Data were expressed as mean ± SEM ( n = 3 per group; * p < 0.05). In panels (A) and (B), immunoblots are representative of the mean value. (C) Proposed model by which NEDD4‐L regulates autophagy via obestatin signaling under dystrophic conditions. Tyrosine switch on NEDD4‐L activates autoubiquitination that serves as a scaffold to recruit the deubiquitinase enzyme USP10 to form a deubiquitination complex, which stabilizes VPS34 to promote autophagy through the removal of the ubiquitin chains on VPS34 and activation of Beclin1 complex. In parallel, NEDD4‐L favors AMPK exposure to CaMKKß and consequent activation of ULK1. Under DMD conditions, AMPK does not inhibit mTORC1, but sustains ULK1/Beclin1 activity and autophagy. In nonpathological conditions, lack of action of non‐RTK on NEDD4‐L favors interaction between c‐Src and NEDD4‐L, thus impairing the VPS34 stabilization and AMPK activation.

Article Snippet: To knockdown NEDD4, AMPKα or USP10 expression, siRNA specifically targeting human NEDD4‐L (sc‐75894; Santa Cruz Biotechnology, CA, USA), AMPKα (sc‐45312; Santa Cruz Biotechnology), or USP10 (sc‐76811; Santa Cruz Biotechnology) was used. siRNA duplexes targeting preproghrelin or c‐Src were selected from ON‐TARGET plus SMARTpool siRNA from Thermo Fisher Scientific (Dharmacon, CO, USA). shRNA‐mediated gene silencing was used for ULK1 knockdown (sc‐44182‐SH; Santa Cruz Biotechnology) in DMD cells.

Techniques: Activation Assay, Western Blot, Transfection, Ubiquitin Proteomics, Activity Assay

Journal: bioRxiv

Article Title: Rare centenarian SIRT6 variants elevate SIRT6 protein levels and resist cellular senescence

doi: 10.1101/2025.09.29.679208

Figure Lengend Snippet: (A) Schematic representation of the known domains in the SIRT6 protein. The green bar denotes the nuclear localization signal (NLS), orange lines indicate the positions of centenarian SIRT6 mutations, and the yellow bar marks the C-terminal region required for USP10 binding. (B) SIRT6 protein levels in cytoplasmic and nuclear fractions of WT and CENT hMSCs. (C) Volcano plot showing differentially interacting proteins between WT SIRT6 and CentSIRT6 in hMSCs, identified by endogenous SIRT6 IP-MS. Protein abundances were normalized to SIRT6 levels in each pull-down sample. (D) Endogenous SIRT6 was immunoprecipitated from WT and CENT hMSC lysates using anti-SIRT6 antibody (D8D12). IP samples and input lysates were analyzed by western blotting with antibodies against SIRT6 (2G1H1) and Vimentin (VIM). Input represents whole-cell lysate prior to immunoprecipitation. (E) Protein levels of Vimentin and SIRT6 in WT and CENT hMSCs transfected with siRNAs targeting Vimentin. Protein levels were normalized to β-Actin. (F) Protein levels of Flag-VIM and SIRT6 in WT and CENT hMSCs transduced with lentiviruses expressing Flag or Flag-VIM. Protein levels were normalized to β-Actin. (G) Predicted interaction between the VIM IDR and the C-terminal IDR of SIRT6 using FINCHES. The right panel showing the effects of different SIRT6 mutations on these predicted interactions.

Article Snippet: siRNAs targeting human USP10 (sc-365828) and Vimentin (sc-373717) were purchased from Santa Cruz Biotechnology. siRNAs were transfected into MSCs using Lipofectamine RNAiMAX Reagent (Thermo Fisher) according to the manufacturer’s instructions.

Techniques: Binding Assay, Protein-Protein interactions, Immunoprecipitation, Western Blot, Transfection, Transduction, Expressing

Journal: bioRxiv

Article Title: Rare centenarian SIRT6 variants elevate SIRT6 protein levels and resist cellular senescence

doi: 10.1101/2025.09.29.679208

Figure Lengend Snippet: (A) Protein levels of USP10 and SIRT6 in WT and CENT hMSCs transfected with siRNAs targeting USP10. (B) AlphaFold-predicted structural confidence for SIRT6 and VIM. Low confidence scores correspond to predicted intrinsic disorder regions (IDRs).

Article Snippet: siRNAs targeting human USP10 (sc-365828) and Vimentin (sc-373717) were purchased from Santa Cruz Biotechnology. siRNAs were transfected into MSCs using Lipofectamine RNAiMAX Reagent (Thermo Fisher) according to the manufacturer’s instructions.

Techniques: 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 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