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

Journal: Cell Death & Disease

Article Title: RHBDL2 promotes the proliferation, migration, and invasion of pancreatic cancer by stabilizing the N1ICD via the OTUD7B and activating the Notch signaling pathway

doi: 10.1038/s41419-022-05379-3

Figure Lengend Snippet: A The deubiquitinase screening procedure for N1ICD. B Western blot analysis was performed to measure the expression levels of N1ICD, OTUD7B, ATXN3, and USP10 in the indicated PC cells. Immunoprecipitation assay of OTUD7B and N1ICD ( C–D ), OTUD7B and RHBDL2 ( E–F ). G The expression levels of HA-OTUD7B and Flag-RHBDL2 were confirmed by immunoprecipitation. H The levels of N1ICD and Flag-OTUD7B were determined by Western blot assay. I The PC cells were treated with 15 μM of MG132 for 6 h prior to harvest. The cell lysate was immunoprecipitated with Myc-tag antibody and immunoblotted as indicated. J The levels of N1ICD, Flag-RHBDL2, and OTUD7B in the indicated PC cells were detected by Western blot assay. K The PC cells were treated with 15 μM of MG132 for 6 h prior to harvest. The cell lysate was immunoprecipitated with Myc-tag antibody and immunoblotted as indicated. L The expression levels of N1ICD, RHBDL2, and OTUD7B in the indicated PC cells were determined by Western blot assay.

Article Snippet: Small interfering RNAs (siRNAs) targeting RHBDL2, ATXN3, OTUD7B, and USP10 were acquired from RiboBio (Guangzhou, China).

Techniques: Western Blot, Expressing, Immunoprecipitation

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: Aging (Albany NY)

Article Title: USP10 deletion inhibits macrophage-derived foam cell formation and cellular-oxidized low density lipoprotein uptake by promoting the degradation of CD36

doi: 10.18632/aging.104003

Figure Lengend Snippet: USP10 mediates lipid uptake by macrophage. Cells were treated with Spautin-1 for 24 h or USP10 siRNA for 48 h. Dil-oxLDL was added for the additional 6 h. DAPI for cell nucleus. The images were taken by confocal microscopy ( A , C ) and by flow cytometry ( E , G ). The fluorescence intensity was performed using Image pro plus ( B , D ) and quantitative analysis was showed ( F , H ). *p<0.05, **p<0.01, ***p<0.001 versus each vehicle control. DM:DMSO.

Article Snippet: Control (SC-37007) and USP10 (SC-76811) siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Confocal Microscopy, Flow Cytometry, Fluorescence, Control

Journal: Aging (Albany NY)

Article Title: USP10 deletion inhibits macrophage-derived foam cell formation and cellular-oxidized low density lipoprotein uptake by promoting the degradation of CD36

doi: 10.18632/aging.104003

Figure Lengend Snippet: USP10 inhibition or deletion diminishes the formation of foam cell. ( A , C ) Cells were treated with Spautin-1 or USP10 siRNA and oxLDL (50 μg/ml) for the indicated time. Cell was stained with oil red o. The represent images were showed from three independent experiments. ( B , D ) The number of stained cells were counted. *p<0.05, **p<0.01 versus each vehicle control.

Article Snippet: Control (SC-37007) and USP10 (SC-76811) siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Staining, Control

Journal: Aging (Albany NY)

Article Title: USP10 deletion inhibits macrophage-derived foam cell formation and cellular-oxidized low density lipoprotein uptake by promoting the degradation of CD36

doi: 10.18632/aging.104003

Figure Lengend Snippet: USP10 regulates the protein expression of CD36. ( A , C ) THP1 and RAW264.7 cells were exposed to Spautin-1 or USP10 siRNA. Protein was extracted and subjected to western blot for expression of CD36. ( B , D ) The quantitation of CD36 band were performed. ( E , F ) The treated cell posted with Spautin-1 was stained with FITC-labeled CD36 antibody followed by flow cytometry. ( G ) Cells were stained with CD36 and Cy3-conjugated antibody and subjected to confocal microscopy. *p<0.05, **p<0.01, ***p<0.001 versus each vehicle control.

Article Snippet: Control (SC-37007) and USP10 (SC-76811) siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Quantitation Assay, Staining, Labeling, Flow Cytometry, Confocal Microscopy, Control

Journal: Aging (Albany NY)

Article Title: USP10 deletion inhibits macrophage-derived foam cell formation and cellular-oxidized low density lipoprotein uptake by promoting the degradation of CD36

doi: 10.18632/aging.104003

Figure Lengend Snippet: OxLDL-induced the upregulation of CD36 is reduced by USP10 inhibition. ( A ) Cells were treated with Spautin-1/USP10 siRNA, oxLDL or the combination of the two treatments. Protein was harvested for western blot assay to test CD36 expression. ( B ) The band of CD36 was counted. ( C , D ) The treated cells posted with Spautin-1 were stained FITC-CD36 antibody followed by flow cytometry. ( E ) THP1 cell was incubated with CD36 antibody and DAPI for cell nucleus. *p<0.05, ***p<0.001 versus each vehicle control.

Article Snippet: Control (SC-37007) and USP10 (SC-76811) siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Western Blot, Expressing, Staining, Flow Cytometry, Incubation, Control

Journal: Aging (Albany NY)

Article Title: USP10 deletion inhibits macrophage-derived foam cell formation and cellular-oxidized low density lipoprotein uptake by promoting the degradation of CD36

doi: 10.18632/aging.104003

Figure Lengend Snippet: USP10 stabilizes CD36. ( A , B ) THP1 cells were treated with CHX or CHX+Spautin-1/USP10 siRNA for 0, 12, 24 and 36 h. CD36 expression was detected using western blot assay. ( C ) And then the band of CD36 was calculated. ( D , E ) Cells were treated with Spautin-1, MG132. Western blot was performed to test the expression of CD36. ( F ) Total RNA was extracted and RT-qPCR was performed to detected mRNA level of CD36. *p<0.05 versus Spautin-1 treatment.

Article Snippet: Control (SC-37007) and USP10 (SC-76811) siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Aging (Albany NY)

Article Title: USP10 deletion inhibits macrophage-derived foam cell formation and cellular-oxidized low density lipoprotein uptake by promoting the degradation of CD36

doi: 10.18632/aging.104003

Figure Lengend Snippet: USP10 interacts with CD36. ( A , B ) Protein lysates were extracted from THP1 cells and subjected to Co-IP assay. ( C ) Cells were transfected with FLAG-tagged USP10 for 48 h and then stained with FLAG and CD36 antibodies, followed by confocal assay. ( D , E ) Cells were treated with Spautin-1 or USP10 siRNA. Immunoprecipitated with CD36 beads was subjected to immunoblotted for Ub and K48.

Article Snippet: Control (SC-37007) and USP10 (SC-76811) siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Co-Immunoprecipitation Assay, Transfection, Staining, Confocal Assay, Immunoprecipitation

Journal: Aging (Albany NY)

Article Title: USP10 deletion inhibits macrophage-derived foam cell formation and cellular-oxidized low density lipoprotein uptake by promoting the degradation of CD36

doi: 10.18632/aging.104003

Figure Lengend Snippet: USP10-mediated lipid uptake depends on CD36 expression. ( A ) Macrophage was transfected with FLAG-USP10 plasmid with different doses for 48 h, followed by Dil-oxLDL for 6 h. ( B ) Macrophage was treated with Spautin-1 and /or FLAG-CD36 plasmid for 48 h. Dil-oxLDL was added for the additional 6 h. DAPI for cell nucleus. The images were taken by confocal microscopy.

Article Snippet: Control (SC-37007) and USP10 (SC-76811) siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Confocal Microscopy

Journal: Aging (Albany NY)

Article Title: USP10 deletion inhibits macrophage-derived foam cell formation and cellular-oxidized low density lipoprotein uptake by promoting the degradation of CD36

doi: 10.18632/aging.104003

Figure Lengend Snippet: A proposed mechanism for USP10 to regulate foam cell formation via stabilizing the CD36 level.

Article Snippet: Control (SC-37007) and USP10 (SC-76811) siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: