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Cell Signaling Technology Inc usp10 specific sirna
Usp10 Specific Sirna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/usp10+specific+sirna/SignalSilence+USP10+siRNA+I/pmc04505586-421-13-15
Average 85 stars, based on 1 article reviews

usp10 specific sirna - by Bioz Stars, 2026-10

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Transfection:

Article Title: TRAF Family Member-associated NF-κB Activator (TANK) Inhibits Genotoxic Nuclear Factor κB Activation by Facilitating Deubiquitinase USP10-dependent Deubiquitination of TRAF6 Ligase
Article Snippet: .. A , HEK293T cells were transfected with control siRNA ( siCtrl ) or USP10-specific siRNA (CST#7747). ..

Article Title: TRAF Family Member-associated NF-κB Activator (TANK) Inhibits Genotoxic Nuclear Factor κB Activation by Facilitating Deubiquitinase USP10-dependent Deubiquitination of TRAF6 Ligase
Article Snippet: .. A, HEK293T cells were transfected with control siRNA or USP10-specific siRNA (CST#7747). ..

Control:

Article Title: TRAF Family Member-associated NF-κB Activator (TANK) Inhibits Genotoxic Nuclear Factor κB Activation by Facilitating Deubiquitinase USP10-dependent Deubiquitination of TRAF6 Ligase
Article Snippet: .. A, HEK293T cells were transfected with control siRNA or USP10-specific siRNA (CST#7747). ..

Image Search Results

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.

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

USP10-KD enhances dopamine-induced apoptosis. A , SH-SY5Y cells were transfected with USP10-siRNA (USP10-1, 2, or 3) or control (NT) using Lipofectamine RNAiMAX. Whole cell lysates prepared from transfected cells were characterized by Western blotting with anti-USP10 and anti-β-actin antibodies. B , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-1 or 2) or control (siNT) using Lipofectamine RNAiMAX. The cells were then treated with 0.1 to 0.4 mM dopamine or DMSO for 12 h. The cells were treated with CCK-8 solution for 1 h. Culture supernatant was prepared from the transfected cells, and the absorbance (485 nm) of the culture supernatant was measured with an absorption meter (TriStar LB 941). The ratio of absorbance of cells to that of control siRNA (NT) treated with DMSO was presented as the mean and standard deviation (SD) of three samples. The significance of the differences was assessed by a one-way ANOVA, followed by Tukey's multiple comparison test. ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. C and D , USP10-KD (siUSP10-1 or 2) and control (siNT) cells were treated with 0.4 mM dopamine or DMSO for 4 and 8 h, and cells were then stained with anticleavage caspase-3 ( red ) and Hoechst 33258 ( blue ). The staining was evaluated with a fluorescence microscope. The ratio of cells with cleaved caspase-3 relative to total cells (measured by number of nuclei) was presented as the mean and SD of five samples in ( D ). The significance of the difference in <xref ref-type=

Journal: The Journal of Biological Chemistry

Article Title: USP10 inhibits the dopamine-induced reactive oxygen species–dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity

doi: 10.1016/j.jbc.2021.101448

Figure Lengend Snippet: USP10-KD enhances dopamine-induced apoptosis. A , SH-SY5Y cells were transfected with USP10-siRNA (USP10-1, 2, or 3) or control (NT) using Lipofectamine RNAiMAX. Whole cell lysates prepared from transfected cells were characterized by Western blotting with anti-USP10 and anti-β-actin antibodies. B , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-1 or 2) or control (siNT) using Lipofectamine RNAiMAX. The cells were then treated with 0.1 to 0.4 mM dopamine or DMSO for 12 h. The cells were treated with CCK-8 solution for 1 h. Culture supernatant was prepared from the transfected cells, and the absorbance (485 nm) of the culture supernatant was measured with an absorption meter (TriStar LB 941). The ratio of absorbance of cells to that of control siRNA (NT) treated with DMSO was presented as the mean and standard deviation (SD) of three samples. The significance of the differences was assessed by a one-way ANOVA, followed by Tukey's multiple comparison test. ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. C and D , USP10-KD (siUSP10-1 or 2) and control (siNT) cells were treated with 0.4 mM dopamine or DMSO for 4 and 8 h, and cells were then stained with anticleavage caspase-3 ( red ) and Hoechst 33258 ( blue ). The staining was evaluated with a fluorescence microscope. The ratio of cells with cleaved caspase-3 relative to total cells (measured by number of nuclei) was presented as the mean and SD of five samples in ( D ). The significance of the difference in Figure 1 D was assessed by Brown–Forsythe and Welch ANOVA followed by Dunnett's T3 multiple comparisons test. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Scale bar is 10 μm. ANOVA, analysis of variance; NS, Not significant; USP10, ubiquitin-specific protease 10.

Article Snippet: Small interfering RNAs (siRNAs) specific to human USP10 RNA (Oligo ID: HSS113446, HSS113447), p62 RNA (Oligo ID: HSS113116, HSS113117), Nrf2 RNA (Oligo ID: HSS107130, HSS181505, HSS18506), and the negative control siRNA (Cat. No. 12935-100) were purchased from Thermo Fisher Scientific. siRNA specific to Keap1 RNA (Oligo ID: SI03246439, SI04288844, SI04267886, SI04155424) and the negative control siRNA (Cat. No. 1027280) were purchased from Qiagen.

Techniques: Transfection, Control, Western Blot, CCK-8 Assay, Standard Deviation, Comparison, Staining, Fluorescence, Microscopy, Ubiquitin Proteomics

USP10-KD augments dopamine-induced ROS production and ROS-dependent cell death. A and B , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-2) or control (siNT) using Lipofectamine RNAiMAX. Cells were treated with 5 μM CM-H2DCFDA ( Green ), an ROS-sensitive fluorescence dye, for 30 min. Transfected cells were then treated with 0.4 mM dopamine or DMSO for 6 h. The fluorescence intensity was measured. The ratio of fluorescence intensity of cells relative to that of the control (siNT) treated with DMSO was presented as the mean and SD from three samples. ∗ p < 0.05; ∗∗∗∗ p < 0.0001. Scale bar is 10 μm. Bright-field observations of cells were also presented. C , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-1 or 2) or control (siNT) using Lipofectamine RNAiMAX. Cells were pretreated with 500 μM N-acetyl cysteine 30 min before dopamine treatment and further treated with 0.2 to 0.4 mM dopamine or DMSO for 12 h. Cells were treated with CCK-8 solution for 1 h. Cell viability was evaluated by measuring the absorbance (485 nm) of culture medium with an absorbance meter (TriStar LB 941). The ratio of absorbance obtained from cells relative to that of the control (siNT) treated with DMSO was presented as the mean and SD from three samples. ∗∗∗∗ p < 0.0001. D , USP10-KD (siUSP10-2) and control (siNT) cells were treated with 50 to 200 μM H 2 O 2 for 12 h. Cell viability was measured with the CCK-8 kit. The absorbance obtained from cells was normalized to that of the control (siNT) treated with DMSO, and the ratio was presented as the mean and SD from three samples. ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. NS, not significant; USP10, ubiquitin-specific protease 10.

Journal: The Journal of Biological Chemistry

Article Title: USP10 inhibits the dopamine-induced reactive oxygen species–dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity

doi: 10.1016/j.jbc.2021.101448

Figure Lengend Snippet: USP10-KD augments dopamine-induced ROS production and ROS-dependent cell death. A and B , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-2) or control (siNT) using Lipofectamine RNAiMAX. Cells were treated with 5 μM CM-H2DCFDA ( Green ), an ROS-sensitive fluorescence dye, for 30 min. Transfected cells were then treated with 0.4 mM dopamine or DMSO for 6 h. The fluorescence intensity was measured. The ratio of fluorescence intensity of cells relative to that of the control (siNT) treated with DMSO was presented as the mean and SD from three samples. ∗ p < 0.05; ∗∗∗∗ p < 0.0001. Scale bar is 10 μm. Bright-field observations of cells were also presented. C , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-1 or 2) or control (siNT) using Lipofectamine RNAiMAX. Cells were pretreated with 500 μM N-acetyl cysteine 30 min before dopamine treatment and further treated with 0.2 to 0.4 mM dopamine or DMSO for 12 h. Cells were treated with CCK-8 solution for 1 h. Cell viability was evaluated by measuring the absorbance (485 nm) of culture medium with an absorbance meter (TriStar LB 941). The ratio of absorbance obtained from cells relative to that of the control (siNT) treated with DMSO was presented as the mean and SD from three samples. ∗∗∗∗ p < 0.0001. D , USP10-KD (siUSP10-2) and control (siNT) cells were treated with 50 to 200 μM H 2 O 2 for 12 h. Cell viability was measured with the CCK-8 kit. The absorbance obtained from cells was normalized to that of the control (siNT) treated with DMSO, and the ratio was presented as the mean and SD from three samples. ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. NS, not significant; USP10, ubiquitin-specific protease 10.

Techniques: Transfection, Control, Fluorescence, CCK-8 Assay, Ubiquitin Proteomics

$USP10 regulates the antioxidant Nrf2/Keap1/p62 pathway in dopamine-treated neuronal cells. A , SH-SY5Y cells were transfected with USP10-siRNA (USP10-1 or 2) or control (NT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 and 8 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. B , SH-SY5Y cells were pretreated with 0.3 mM ascorbic acid 30 min before dopamine treatment and then treated with 0.2 to 0.4 mM dopamine or DMSO for 8 h. Whole cell lysates prepared from these cells were characterized by Western blotting using the indicated antibodies. C , SH-SY5Y cells were treated with 25 to 200 μM H 2 O 2 or 0.4 mM dopamine for 8 h. Whole cell lysates prepared from these cells were characterized by Western blotting using the indicated antibodies. D , SH-SY5Y cells were transfected with USP10-siRNA (USP10-1 or 2) or control (NT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 h. Lysates of nuclear and cytoplasmic fractions were prepared from transfected cells and characterized by Western blotting using the indicated antibodies. α-tubulin and HDAC1 were used as protein markers localized in the cytoplasm and nucleus, respectively. USP10, ubiquitin-specific protease 10.$

Journal: The Journal of Biological Chemistry

Article Title: USP10 inhibits the dopamine-induced reactive oxygen species–dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity

doi: 10.1016/j.jbc.2021.101448

Figure Lengend Snippet: USP10 regulates the antioxidant Nrf2/Keap1/p62 pathway in dopamine-treated neuronal cells. A , SH-SY5Y cells were transfected with USP10-siRNA (USP10-1 or 2) or control (NT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 and 8 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. B , SH-SY5Y cells were pretreated with 0.3 mM ascorbic acid 30 min before dopamine treatment and then treated with 0.2 to 0.4 mM dopamine or DMSO for 8 h. Whole cell lysates prepared from these cells were characterized by Western blotting using the indicated antibodies. C , SH-SY5Y cells were treated with 25 to 200 μM H 2 O 2 or 0.4 mM dopamine for 8 h. Whole cell lysates prepared from these cells were characterized by Western blotting using the indicated antibodies. D , SH-SY5Y cells were transfected with USP10-siRNA (USP10-1 or 2) or control (NT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 h. Lysates of nuclear and cytoplasmic fractions were prepared from transfected cells and characterized by Western blotting using the indicated antibodies. α-tubulin and HDAC1 were used as protein markers localized in the cytoplasm and nucleus, respectively. USP10, ubiquitin-specific protease 10.

Techniques: Transfection, Control, Western Blot, Ubiquitin Proteomics

Nrf2-KD and p62-KD enhance dopamine-induced cell death. A and B , SH-SY5Y cells were transfected with Nrf2-siRNA (Nrf2-1, 2, or 3) or control (NT) by Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. C , SH-SY5Y cells were transfected with Nrf2-siRNA (siNrf2-1, 2 or 3) or control (siNT) by Lipofectamine RNAiMAX. SH-SY5Y cells were treated with 0.2 to 0.4 mM dopamine or DMSO for 12 h. Cells were treated with CCK-8 solution for 1 h. Culture medium was prepared from transfected cells, and the absorbance (485 nm) of culture medium was measured by an absorbance meter (TriStar LB 941). The ratio of absorbance of the cells relative to that of the control siRNA (siNT) treated with DMSO was presented as the mean and SD from three samples. ∗∗∗∗ p < 0.0001. NS, not significant. D , SH-SY5Y cells were transfected with p62-siRNA (p62-1 or 3) or control (NT) using Lipofectamine RNAiMAX. Cells were then treated with 0.4 mM dopamine or DMSO for 4 h or 8 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. E , SH-SY5Y cells were transfected with p62-siRNA (sip62-1 or 2), USP10-siRNA (siUSP10-1 or 2) and the control (siNT) using Lipofectamine RNAiMAX. Cells were then treated with 0.4 mM dopamine or DMSO for 12 h and with CCK-8 solution for 1 h, and the cell viability was measured with the CCK-8 kit by measuring the absorbance (485 nm) of culture supernatant. The ratio of the absorbance of cells relative to that of the control (siNT) treated with DMSO was presented as the mean and SD from three samples. ∗∗∗∗ p < 0.0001.

Journal: The Journal of Biological Chemistry

Article Title: USP10 inhibits the dopamine-induced reactive oxygen species–dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity

doi: 10.1016/j.jbc.2021.101448

Figure Lengend Snippet: Nrf2-KD and p62-KD enhance dopamine-induced cell death. A and B , SH-SY5Y cells were transfected with Nrf2-siRNA (Nrf2-1, 2, or 3) or control (NT) by Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. C , SH-SY5Y cells were transfected with Nrf2-siRNA (siNrf2-1, 2 or 3) or control (siNT) by Lipofectamine RNAiMAX. SH-SY5Y cells were treated with 0.2 to 0.4 mM dopamine or DMSO for 12 h. Cells were treated with CCK-8 solution for 1 h. Culture medium was prepared from transfected cells, and the absorbance (485 nm) of culture medium was measured by an absorbance meter (TriStar LB 941). The ratio of absorbance of the cells relative to that of the control siRNA (siNT) treated with DMSO was presented as the mean and SD from three samples. ∗∗∗∗ p < 0.0001. NS, not significant. D , SH-SY5Y cells were transfected with p62-siRNA (p62-1 or 3) or control (NT) using Lipofectamine RNAiMAX. Cells were then treated with 0.4 mM dopamine or DMSO for 4 h or 8 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. E , SH-SY5Y cells were transfected with p62-siRNA (sip62-1 or 2), USP10-siRNA (siUSP10-1 or 2) and the control (siNT) using Lipofectamine RNAiMAX. Cells were then treated with 0.4 mM dopamine or DMSO for 12 h and with CCK-8 solution for 1 h, and the cell viability was measured with the CCK-8 kit by measuring the absorbance (485 nm) of culture supernatant. The ratio of the absorbance of cells relative to that of the control (siNT) treated with DMSO was presented as the mean and SD from three samples. ∗∗∗∗ p < 0.0001.

Techniques: Transfection, Control, Western Blot, CCK-8 Assay

Keap1-KD attenuates USP10-KD/dopamine-induced cell death. A , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-2), Keap1-siRNA (siKeap1-5, 6, or 7) or control (siNT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 8 h. Whole cell lysates were characterized by Western blotting using the indicated antibodies. B , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-2), Keap1-siRNA (siKeap1-5, 6 or 7), or control (siNT) using Lipofectamine RNAiMAX. Cells were then treated with 0.2 to 0.4 mM dopamine or DMSO for 12 h and with CCK-8 solution for 1 h, and the cell viability was measured with the CCK-8 kit by measuring the absorbance (485 nm) of the culture medium. The ratio of the absorbance of the cells to that of the control cells (siNT) treated with DMSO was presented as the mean and SD of three samples. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. USP10, ubiquitin-specific protease 10.

Journal: The Journal of Biological Chemistry

Article Title: USP10 inhibits the dopamine-induced reactive oxygen species–dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity

doi: 10.1016/j.jbc.2021.101448

Figure Lengend Snippet: Keap1-KD attenuates USP10-KD/dopamine-induced cell death. A , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-2), Keap1-siRNA (siKeap1-5, 6, or 7) or control (siNT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 8 h. Whole cell lysates were characterized by Western blotting using the indicated antibodies. B , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-2), Keap1-siRNA (siKeap1-5, 6 or 7), or control (siNT) using Lipofectamine RNAiMAX. Cells were then treated with 0.2 to 0.4 mM dopamine or DMSO for 12 h and with CCK-8 solution for 1 h, and the cell viability was measured with the CCK-8 kit by measuring the absorbance (485 nm) of the culture medium. The ratio of the absorbance of the cells to that of the control cells (siNT) treated with DMSO was presented as the mean and SD of three samples. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. USP10, ubiquitin-specific protease 10.

Techniques: Transfection, Control, Western Blot, CCK-8 Assay, Ubiquitin Proteomics

Dopamine enhances PERK phosphorylation. A , SH-SY5Y cells were pretreated with 1 μM GSK260614 (PERK inhibitor) or DMSO 30 min before dopamine treatment and then treated with 0.2 to 0.4 mM dopamine or DMSO for 8 h. In addition, cells were treated with 1 μM thapsigargin (Tg) for 30 min, and this was used as positive control for PERK phosphorylation. Whole cell lysates prepared from these cells were characterized by Western blotting using the indicated antibodies. B , SH-SY5Y cells were transfected with USP10-siRNA (USP10-1 or 2) or control (NT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 and 8 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. USP10, ubiquitin-specific protease 10.

Journal: The Journal of Biological Chemistry

Article Title: USP10 inhibits the dopamine-induced reactive oxygen species–dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity

doi: 10.1016/j.jbc.2021.101448

Figure Lengend Snippet: Dopamine enhances PERK phosphorylation. A , SH-SY5Y cells were pretreated with 1 μM GSK260614 (PERK inhibitor) or DMSO 30 min before dopamine treatment and then treated with 0.2 to 0.4 mM dopamine or DMSO for 8 h. In addition, cells were treated with 1 μM thapsigargin (Tg) for 30 min, and this was used as positive control for PERK phosphorylation. Whole cell lysates prepared from these cells were characterized by Western blotting using the indicated antibodies. B , SH-SY5Y cells were transfected with USP10-siRNA (USP10-1 or 2) or control (NT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 and 8 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. USP10, ubiquitin-specific protease 10.

Techniques: Phospho-proteomics, Positive Control, Western Blot, Transfection, Control, Ubiquitin Proteomics

USP10-KD does not reduce the amount of Nrf2 mRNA in neuronal cells. A , SH-SY5Y cells were transfected with USP10-siRNA (USP10-2) or control (NT) using Lipofectamine RNAiMAX. Cells were pretreated with 5 nM Bafilomycin A1 30 min before dopamine treatment and further treated with 0.4 mM dopamine or DMSO with or without 5 μM MG-132 for 8 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. B , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-2) or control (siNT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 3 or 6 h. Total RNA was extracted from these cells, and the relative amount of NQO1, GSTM1, p62, and Nrf2 mRNA to β-actin mRNA was measured by real-time RT-PCR. The data were presented as the means ± SD (n = 3). ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. NS, Not significant; USP10, ubiquitin-specific protease 10.

Journal: The Journal of Biological Chemistry

Article Title: USP10 inhibits the dopamine-induced reactive oxygen species–dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity

doi: 10.1016/j.jbc.2021.101448

Figure Lengend Snippet: USP10-KD does not reduce the amount of Nrf2 mRNA in neuronal cells. A , SH-SY5Y cells were transfected with USP10-siRNA (USP10-2) or control (NT) using Lipofectamine RNAiMAX. Cells were pretreated with 5 nM Bafilomycin A1 30 min before dopamine treatment and further treated with 0.4 mM dopamine or DMSO with or without 5 μM MG-132 for 8 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. B , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-2) or control (siNT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 3 or 6 h. Total RNA was extracted from these cells, and the relative amount of NQO1, GSTM1, p62, and Nrf2 mRNA to β-actin mRNA was measured by real-time RT-PCR. The data were presented as the means ± SD (n = 3). ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. NS, Not significant; USP10, ubiquitin-specific protease 10.

Techniques: Transfection, Control, Western Blot, Quantitative RT-PCR, Ubiquitin Proteomics

USP10-KD suppresses global translation in dopamine-treated cells. A , SH-SY5Y cells were transfected with USP10-siRNA (USP10-1 or 2) or control (NT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 or 8 h and further treated with 10 μg/ml puromycin for 10 min. Cell lysates were characterized by Western blotting using the indicated antibodies. B and C , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-1 or 2) or control (siNT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 or 8 h and then stained with anti-G3BP1 ( green ) and anti-TIA1 ( red ) antibody. Nuclei were counterstained using Hoechst 33258 ( blue ). The proportions of cells containing G3BP1/TIA1-SG were presented as the mean and SD from three samples in ( C ). ∗∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. Arrowheads indicate cells with G3BP1/TIA1-double-positive SG. Scale bar is 10 μm. D and E , SH-SY5Y cells were treated with 0.4 mM dopamine for 8 h and then stained with anti-G3BP1 ( green ) and anti-USP10 ( red ) antibodies. Nuclei were counterstained using Hoechst 33258 ( blue ). Scale bar is 10 μm. The proportions of cells containing G3BP1/USP10-SG or G3BP-1-SG were presented as the mean and SD from three samples in ( E ). USP10, ubiquitin-specific protease 10.

Journal: The Journal of Biological Chemistry

Article Title: USP10 inhibits the dopamine-induced reactive oxygen species–dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity

doi: 10.1016/j.jbc.2021.101448

Figure Lengend Snippet: USP10-KD suppresses global translation in dopamine-treated cells. A , SH-SY5Y cells were transfected with USP10-siRNA (USP10-1 or 2) or control (NT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 or 8 h and further treated with 10 μg/ml puromycin for 10 min. Cell lysates were characterized by Western blotting using the indicated antibodies. B and C , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-1 or 2) or control (siNT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 or 8 h and then stained with anti-G3BP1 ( green ) and anti-TIA1 ( red ) antibody. Nuclei were counterstained using Hoechst 33258 ( blue ). The proportions of cells containing G3BP1/TIA1-SG were presented as the mean and SD from three samples in ( C ). ∗∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. Arrowheads indicate cells with G3BP1/TIA1-double-positive SG. Scale bar is 10 μm. D and E , SH-SY5Y cells were treated with 0.4 mM dopamine for 8 h and then stained with anti-G3BP1 ( green ) and anti-USP10 ( red ) antibodies. Nuclei were counterstained using Hoechst 33258 ( blue ). Scale bar is 10 μm. The proportions of cells containing G3BP1/USP10-SG or G3BP-1-SG were presented as the mean and SD from three samples in ( E ). USP10, ubiquitin-specific protease 10.

Techniques: Transfection, Control, Western Blot, Staining, Ubiquitin Proteomics

USP10-KD induces ubiquitination of ribosomal proteins. SH-SY5Y cells were transfected with USP10-siRNA (USP10-1 or 2) or control (NT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 and 8 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. The experiments in <xref ref-type=

Figures 6 B and were performed simultaneously as one experiment. Therefore, the data for β-actin and USP10 shown in Figure 6 B are also represented in Figure 9 . USP10, ubiquitin-specific protease 10. " width="100%" height="100%">

Journal: The Journal of Biological Chemistry

Article Title: USP10 inhibits the dopamine-induced reactive oxygen species–dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity

doi: 10.1016/j.jbc.2021.101448

Figure Lengend Snippet: USP10-KD induces ubiquitination of ribosomal proteins. SH-SY5Y cells were transfected with USP10-siRNA (USP10-1 or 2) or control (NT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 and 8 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. The experiments in Figures 6 B and were performed simultaneously as one experiment. Therefore, the data for β-actin and USP10 shown in Figure 6 B are also represented in Figure 9 . USP10, ubiquitin-specific protease 10.

Techniques: Ubiquitin Proteomics, Transfection, Control, Western Blot

USP10-KD reduces the amount of phosphorylated p62. A , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-2) or control (siNT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 8 h. Cell lysates prepared from transfected cells were immunoprecipitated with anti-p62 antibody or normal rabbit IgG. Cell lysates (input) and immunoprecipitates (IP) were characterized by Western blotting using the indicated antibodies. B , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-2) or control (siNT) using Lipofectamine RNAiMAX. The cells were pretreated with 5 nM BafA1 30 min before dopamine treatment and further treated with 0.4 mM dopamine or DMSO for 8 h. Cell lysates were characterized by Western blotting using the indicated antibodies. USP10, ubiquitin-specific protease 10.

Journal: The Journal of Biological Chemistry

Article Title: USP10 inhibits the dopamine-induced reactive oxygen species–dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity

doi: 10.1016/j.jbc.2021.101448

Figure Lengend Snippet: USP10-KD reduces the amount of phosphorylated p62. A , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-2) or control (siNT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 8 h. Cell lysates prepared from transfected cells were immunoprecipitated with anti-p62 antibody or normal rabbit IgG. Cell lysates (input) and immunoprecipitates (IP) were characterized by Western blotting using the indicated antibodies. B , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-2) or control (siNT) using Lipofectamine RNAiMAX. The cells were pretreated with 5 nM BafA1 30 min before dopamine treatment and further treated with 0.4 mM dopamine or DMSO for 8 h. Cell lysates were characterized by Western blotting using the indicated antibodies. USP10, ubiquitin-specific protease 10.

Techniques: Transfection, Control, Immunoprecipitation, Western Blot, Ubiquitin Proteomics

USP10-KD reduces p62/Keap1-body formation in dopamine-treated cells. A and B , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-1 or 2) or control (siNT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 8 h and then stained with anti-Keap1 ( green ), anti-phospho-p62 ( green ), and anti-p62 ( red ) antibody. Nuclei were counterstained using Hoechst 33258 ( blue ). The proportions of cells containing p62-body, p62/Keap1-body, or p62/pp62-body were presented as the mean and SD from three samples in ( B ). ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. Scale bar is 10 μm. The arrowheads indicate cells with p62-body, p62/Keap1-body, or p62/pp62-body. C and D , SH-SY5Y cells were transfected with control siRNA using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 8 h and then stained with anti-USP10 ( green ) and anti-p62 ( red ) antibodies. Nuclei were counterstained using Hoechst 33258 ( blue ). Scale bar is 10 μm. The proportions of cells containing p62/USP10-body or p62-body were presented as the mean and SD from three samples in ( D ). USP10, ubiquitin-specific protease 10.

Journal: The Journal of Biological Chemistry

Article Title: USP10 inhibits the dopamine-induced reactive oxygen species–dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity

doi: 10.1016/j.jbc.2021.101448

Figure Lengend Snippet: USP10-KD reduces p62/Keap1-body formation in dopamine-treated cells. A and B , SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-1 or 2) or control (siNT) using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 8 h and then stained with anti-Keap1 ( green ), anti-phospho-p62 ( green ), and anti-p62 ( red ) antibody. Nuclei were counterstained using Hoechst 33258 ( blue ). The proportions of cells containing p62-body, p62/Keap1-body, or p62/pp62-body were presented as the mean and SD from three samples in ( B ). ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. Scale bar is 10 μm. The arrowheads indicate cells with p62-body, p62/Keap1-body, or p62/pp62-body. C and D , SH-SY5Y cells were transfected with control siRNA using Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 8 h and then stained with anti-USP10 ( green ) and anti-p62 ( red ) antibodies. Nuclei were counterstained using Hoechst 33258 ( blue ). Scale bar is 10 μm. The proportions of cells containing p62/USP10-body or p62-body were presented as the mean and SD from three samples in ( D ). USP10, ubiquitin-specific protease 10.

Techniques: Transfection, Control, Staining, Ubiquitin Proteomics

Phosphorylation-mimicking p62 attenuated USP10-KD-induced cell death. A , SH-SY5Y cells stably expressing WT-p62 (W), p62-S349E (E) or p62-S349A (A) or the vector plasmid (V) were established. These SH-SY5Y cells were transfected with USP10-siRNA (USP10-2) or control (NT) by Lipofectamine RNAiMAX. Transfected cells were treated with 0.4 mM dopamine or DMSO for 8 h, and the whole cell lysates were characterized by Western blotting using indicated antibodies. B , the indicated SH-SY5Y cells were transfected with USP10-siRNA (USP10-2) or control (NT) by Lipofectamine RNAiMAX. Transfected cells were treated with 0.4 mM dopamine or DMSO for 12 h. Cells were treated with CCK-8 solution for 1 h, and culture medium was prepared from CCK-8-treated cells. The absorbance (485 nm) of culture medium was then measured with an absorbance meter (TriStar LB 941). The ratio of absorbance obtained from cells relative to that of the control cells (V) transfected with siNT (NT) treated with DMSO was presented as the mean and SD from three samples. ∗∗∗∗ p < 0.0001. C , the indicated SH-SY5Y cells were transfected with USP10-siRNA (USP10-2) or control (NT) by Lipofectamine RNAiMAX. Transfected cells were treated with either 0.4 mM dopamine or DMSO for 8 h, and then the cells were stained with anti-cleaved caspase-3 and Hoechst 33258. The staining was evaluated by a fluorescence microscope. The ratio of cells with cleaved caspase-3 staining relative to total cells measured by the number of nuclei was presented as the mean and SD of seven samples in ( C ). The significance of the differences was assessed by Brown–Forsythe and Welch ANOVA followed by Dunnett's T3 multiple comparisons test. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. D , the current model of USP10 regulation of Nrf2-dependent antioxidant activity in neuronal cells. USP10 activates the antioxidant activity of Nrf2 through two mechanisms. First, USP10 promotes phosphorylation of p62 (pp62-S349) via an unknown molecule (X), such as a phosphatase or kinase. Second, USP10 promotes the dopamine-induced translation of Nrf2 through inhibition of SG formation and/or deubiquitination of ribosomal proteins. NS, Not significant; USP10, ubiquitin-specific protease 10.

Journal: The Journal of Biological Chemistry

Article Title: USP10 inhibits the dopamine-induced reactive oxygen species–dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity

doi: 10.1016/j.jbc.2021.101448

Figure Lengend Snippet: Phosphorylation-mimicking p62 attenuated USP10-KD-induced cell death. A , SH-SY5Y cells stably expressing WT-p62 (W), p62-S349E (E) or p62-S349A (A) or the vector plasmid (V) were established. These SH-SY5Y cells were transfected with USP10-siRNA (USP10-2) or control (NT) by Lipofectamine RNAiMAX. Transfected cells were treated with 0.4 mM dopamine or DMSO for 8 h, and the whole cell lysates were characterized by Western blotting using indicated antibodies. B , the indicated SH-SY5Y cells were transfected with USP10-siRNA (USP10-2) or control (NT) by Lipofectamine RNAiMAX. Transfected cells were treated with 0.4 mM dopamine or DMSO for 12 h. Cells were treated with CCK-8 solution for 1 h, and culture medium was prepared from CCK-8-treated cells. The absorbance (485 nm) of culture medium was then measured with an absorbance meter (TriStar LB 941). The ratio of absorbance obtained from cells relative to that of the control cells (V) transfected with siNT (NT) treated with DMSO was presented as the mean and SD from three samples. ∗∗∗∗ p < 0.0001. C , the indicated SH-SY5Y cells were transfected with USP10-siRNA (USP10-2) or control (NT) by Lipofectamine RNAiMAX. Transfected cells were treated with either 0.4 mM dopamine or DMSO for 8 h, and then the cells were stained with anti-cleaved caspase-3 and Hoechst 33258. The staining was evaluated by a fluorescence microscope. The ratio of cells with cleaved caspase-3 staining relative to total cells measured by the number of nuclei was presented as the mean and SD of seven samples in ( C ). The significance of the differences was assessed by Brown–Forsythe and Welch ANOVA followed by Dunnett's T3 multiple comparisons test. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. D , the current model of USP10 regulation of Nrf2-dependent antioxidant activity in neuronal cells. USP10 activates the antioxidant activity of Nrf2 through two mechanisms. First, USP10 promotes phosphorylation of p62 (pp62-S349) via an unknown molecule (X), such as a phosphatase or kinase. Second, USP10 promotes the dopamine-induced translation of Nrf2 through inhibition of SG formation and/or deubiquitination of ribosomal proteins. NS, Not significant; USP10, ubiquitin-specific protease 10.

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Plasmid Preparation, Transfection, Control, Western Blot, CCK-8 Assay, Staining, Fluorescence, Microscopy, Antioxidant Activity Assay, Inhibition, Ubiquitin Proteomics

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