Journal: Antioxidants

Article Title: PKCiota Inhibits the Ferroptosis of Esophageal Cancer Cells via Suppressing USP14-Mediated Autophagic Degradation of GPX4

doi: 10.3390/antiox13010114

Figure Lengend Snippet: PKCiota regulated the USP14-mediated autophagic degradation of GPX4 in esophageal cancer cells. ( A ) Detection of GPX4 protein level at 48 h after PKCiota siRNA transfection by Western blotting method. ( B , C ) KYSE510 and KYSE450 cells after PKCiota knockdown were treated with CHX (10 μM), analyzed at 0, 3, 6, 9, 12, and 15 h, and immunoblotted for GPX4 and GAPDH. ( D ) Detection of GPX4 expression at 48 h after PKCiota siRNA transfection and MG132 (25 μM)- or BafA1 (100 nM)-added KYSE510 and KYSE450 cells by Western blotting method. ( E ) K63-linkage-specific polyubiquitin antibody was used to detect the K63-linked ubiquitination of GPX4. ( F ) Examination of the interaction among PKCiota, USP14, and GPX4 by co-IP assay. ( G ) Detection of the interaction between PKCiota and GPX4 by PLA technology (Scale bar = 10 μm). ( H ) The binding between GPX4 and USP14 was detected by a co-IP assay. ( I ) The effects of PKCiota silencing on the expression of GPX4, p62, and USP14 were analyzed through Western blotting assay at 48 h after PKCiota siRNA transfection. ( J ) The effects of IU1 (100 μM, 48 h) on GPX4 expression were detected. ( K ) The effects of PKCiota knockdown on phosphorylation at Ser sites of USP14 and ubiquitination of GPX4 at 48 h after PKCiota siRNA transfection were evaluated through Co-IP and Western blotting methods. ( L ) Protein expression of p-AKT, AKT, USP14, and GPX4 after MK2206 (10 μM) treatment was analyzed via Western blotting technology. ( M ) K63-linkage-specific polyubiquitin antibody was used to detect the K63-linked ubiquitination of GPX4. ( N ) Protein level of GPX4 under RSL3 treatment (48 h). ( O ) Effects of PKCiota knockdown and RSL3 treatment on expression of GPX4. ( P , Q ) Cell viability of KYSE510 and KYSE450 cells after PKCiota knockdown and RSL3 treatment (CCK-8 assay). ( R ) Cellular MDA was measured after PKCiota knockdown and RSL3 treatment. ns: not significant; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: Information about the antibodies is as follows: anti-PKCiota antibodies (BD Biosciences, Franklin Lakes, NJ, USA, 610175, 1:1000 dilution; Proteintech, Wuhan, China, 13883-1-AP, 1:1000 dilution), anti-GPX4 antibodies (Abcam, Cambridge, UK, ab125066, 1:2000 dilution; Proteintech, 67763-1-Ig, 1:1000 dilution), anti-USP14 antibodies (Cell signaling technology, Boston, USA, 11931S, 1:1000 dilution; Proteintech, 14517-1-AP, 1:2000 dilution), anti-p62 antibody (MBL, M162-3, 1:1000 dilution), anti-K63 linkage-specific polyubiquitin antibody (Cell Signaling technology, 5621, 1:1000 dilution), anti-p-AKT antibody (Cell Signaling technology, 4060, 1:2000 dilution), anti-AKT antibody (Cell Signaling technology, 4691, 1:1000 dilution), anti-CANX antibody (Proteintech, 10427-2-AP, 1:5000 dilution), anti-SOX2 antibody (Proteintech, 11064-1-AP, 1:1000 dilution), and anti-GAPDH antibodies (Abcam, ab8245, 1:2000 dilution; Proteintech, 10494-1-AP, 1:5000 dilution).

Techniques: Transfection, Western Blot, Expressing, Co-Immunoprecipitation Assay, Binding Assay, CCK-8 Assay

Journal: Antioxidants

Article Title: PKCiota Inhibits the Ferroptosis of Esophageal Cancer Cells via Suppressing USP14-Mediated Autophagic Degradation of GPX4

doi: 10.3390/antiox13010114

Figure Lengend Snippet: PKCiota was regulated by a positive feedback loop and also negatively regulated by miR-145-5p in ESCC. ( A , B ) Effects of USP14 knockdown (48 h after USP14 siRNA transfection) or IU1 (100 μM, 48 h) treatment on the protein levels of PKCiota, GPX4, and USP14 were evaluated by Western blotting. ( C ) Effects of GPX4 knockdown (48 h after GPX4 siRNA transfection) on the expression of PKCiota, USP14, and GPX4 were evaluated by the Western blotting method. ( D ) miR-145-5p expression after transfection (48 h) was detected using qRT-PCR. ( E – I ) Effects of miR-145-5p mimic transfection (48 h) on PKCiota, GPX4, and USP14 were evaluated. ( J ) Effects of miR-145-5p mimic transfection on the colony formation abilities of ESCC cells. ( K ) Effects of miR-145-5p mimic transfection together with RSL3 treatment on expression of GPX4. ( L ) The transcript level of PKCiota under miR-145-5p mimic transfection and RSL3 treatment. ( M , N ) Cell viability of KYSE510 and KYSE450 cells under RSL3 treatment together with miR-145-5p mimic transfection. ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: Information about the antibodies is as follows: anti-PKCiota antibodies (BD Biosciences, Franklin Lakes, NJ, USA, 610175, 1:1000 dilution; Proteintech, Wuhan, China, 13883-1-AP, 1:1000 dilution), anti-GPX4 antibodies (Abcam, Cambridge, UK, ab125066, 1:2000 dilution; Proteintech, 67763-1-Ig, 1:1000 dilution), anti-USP14 antibodies (Cell signaling technology, Boston, USA, 11931S, 1:1000 dilution; Proteintech, 14517-1-AP, 1:2000 dilution), anti-p62 antibody (MBL, M162-3, 1:1000 dilution), anti-K63 linkage-specific polyubiquitin antibody (Cell Signaling technology, 5621, 1:1000 dilution), anti-p-AKT antibody (Cell Signaling technology, 4060, 1:2000 dilution), anti-AKT antibody (Cell Signaling technology, 4691, 1:1000 dilution), anti-CANX antibody (Proteintech, 10427-2-AP, 1:5000 dilution), anti-SOX2 antibody (Proteintech, 11064-1-AP, 1:1000 dilution), and anti-GAPDH antibodies (Abcam, ab8245, 1:2000 dilution; Proteintech, 10494-1-AP, 1:5000 dilution).

Techniques: Transfection, Western Blot, Expressing, Quantitative RT-PCR

Journal: Antioxidants

Article Title: PKCiota Inhibits the Ferroptosis of Esophageal Cancer Cells via Suppressing USP14-Mediated Autophagic Degradation of GPX4

doi: 10.3390/antiox13010114

Figure Lengend Snippet: Identification of hub genes downstream of GPX4 in ESCC. ( A ) GPX4 knockdown was confirmed using Western blotting assay 48 h after GPX4 siRNA transfection. ( B , C ) Venn plot showed the overlapped downregulated and upregulated genes after GPX4 knockdown (si1 and si2). ( D ) GO analysis of DEGs after GPX4 knockdown in KYSE510 cells. ( E ) The PPI network was constructed based on DEGs using the STRING website. ( F ) Module in the PPI was identified using the MCODE plugin in Cytoscape. ( G ) Hub genes were selected through the cytoHubba plugin in Cytoscap (MCC algorithm). ( H ) Transcript levels of CANX, SOX2, and BMP4 after GPX4 knockdown. ( I ) Protein expression of CANX and SOX2 after GPX4 knockdown. ( J ) Protein expression of CANX after PKCiota knockdown. ( K ) Protein expression of USP14 and GPX4 after CANX silence. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: Information about the antibodies is as follows: anti-PKCiota antibodies (BD Biosciences, Franklin Lakes, NJ, USA, 610175, 1:1000 dilution; Proteintech, Wuhan, China, 13883-1-AP, 1:1000 dilution), anti-GPX4 antibodies (Abcam, Cambridge, UK, ab125066, 1:2000 dilution; Proteintech, 67763-1-Ig, 1:1000 dilution), anti-USP14 antibodies (Cell signaling technology, Boston, USA, 11931S, 1:1000 dilution; Proteintech, 14517-1-AP, 1:2000 dilution), anti-p62 antibody (MBL, M162-3, 1:1000 dilution), anti-K63 linkage-specific polyubiquitin antibody (Cell Signaling technology, 5621, 1:1000 dilution), anti-p-AKT antibody (Cell Signaling technology, 4060, 1:2000 dilution), anti-AKT antibody (Cell Signaling technology, 4691, 1:1000 dilution), anti-CANX antibody (Proteintech, 10427-2-AP, 1:5000 dilution), anti-SOX2 antibody (Proteintech, 11064-1-AP, 1:1000 dilution), and anti-GAPDH antibodies (Abcam, ab8245, 1:2000 dilution; Proteintech, 10494-1-AP, 1:5000 dilution).

Techniques: Western Blot, Transfection, Construct, Expressing

Journal: Antioxidants

Article Title: PKCiota Inhibits the Ferroptosis of Esophageal Cancer Cells via Suppressing USP14-Mediated Autophagic Degradation of GPX4

doi: 10.3390/antiox13010114

Figure Lengend Snippet: Expression and prognostic role of PKCiota and GPX4 in ESCC samples. ( A ) Representative IHC images of PKCiota and GPX4 in tumor samples. ( B ) Positive rates of PKCiota and GPX4 in tumors (Scale bar = 50 μm). ( C ) The Kaplan–Meier curve shows the association between lower survival rate and co-expression of PKCiota and GPX4 in ESCC. ( D ) Kaplan–Meier curve showing the association between survival time of ESCC patients and positive expression of PKCiota and USP14 in the GSE53625 dataset. ( E ) The cBioPortal database was used to analyze the mutation to Ser of USP14 and Lys mutation to other amino acids in GPX4 in pan-cancer. ( F ) Schematic of mechanisms underlying PKCiota-induced resistance of ESCC cells to ferroptosis.

Article Snippet: Information about the antibodies is as follows: anti-PKCiota antibodies (BD Biosciences, Franklin Lakes, NJ, USA, 610175, 1:1000 dilution; Proteintech, Wuhan, China, 13883-1-AP, 1:1000 dilution), anti-GPX4 antibodies (Abcam, Cambridge, UK, ab125066, 1:2000 dilution; Proteintech, 67763-1-Ig, 1:1000 dilution), anti-USP14 antibodies (Cell signaling technology, Boston, USA, 11931S, 1:1000 dilution; Proteintech, 14517-1-AP, 1:2000 dilution), anti-p62 antibody (MBL, M162-3, 1:1000 dilution), anti-K63 linkage-specific polyubiquitin antibody (Cell Signaling technology, 5621, 1:1000 dilution), anti-p-AKT antibody (Cell Signaling technology, 4060, 1:2000 dilution), anti-AKT antibody (Cell Signaling technology, 4691, 1:1000 dilution), anti-CANX antibody (Proteintech, 10427-2-AP, 1:5000 dilution), anti-SOX2 antibody (Proteintech, 11064-1-AP, 1:1000 dilution), and anti-GAPDH antibodies (Abcam, ab8245, 1:2000 dilution; Proteintech, 10494-1-AP, 1:5000 dilution).

Techniques: Expressing, Mutagenesis

Journal: Journal of translational medicine

Article Title: Ubiquitin-specific protease 14 targets PFKL-mediated glycolysis to promote the proliferation and migration of oral squamous cell carcinoma.

doi: 10.1186/s12967-024-04943-z

Figure Lengend Snippet: Fig. 1 The upregulation of ubiquitin-specific protease 14 predicts poor clinical outcomes in patients with oral squamous cell carcinoma. A–D USP14 expression of patients with OSCC from the TCGA database. USP14 was aberrantly overexpressed in OSCC tumor tissues compared to normal tissues (A). USP14 expression in OSCC was higher than that in matched normal samples (B). Upregulated expression of USP14 was significantly associated with T stage (C) and N stage (D). E–G Overall survival (OS), progress free interval (PFI) and disease specific survival (DSS) based on USP14 expression in OSCC (TCGA). USP14 high (red) group corresponds to the fourth quartile of expression, while USP14 low (blue) group corresponds to the first quartile. H The protein levels of USP14 in four OSCC cell lines were compared with a normal oral epithelial cell line HOK by Western blotting analysis. I The expression pattern of USP14 was examined in OSCC tissues and their adjacent normal epithelial tissues by immunohistochemical staining (n = 70). *P < 0.05, ***P < 0.001

Article Snippet: Primary antibodies against the following proteins were used for Western blotting analysis: USP14 (cat. no. sc-398009; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Flag (cat. no. 20543-1-AP; 1:2000 dilution; ProteinTech Group, Inc., Chicago, Illinois, USA), HA (cat. no. #2367; 1:1000 dilution; Cell Signaling Technology, Inc., Boston, MA, USA), Ub (cat. no. #3936; 1:1000 dilution; Cell Signaling Technology, Inc., Boston, MA, USA), PFKL (cat. no. ab97443; 1:2000 dilution; Abcam, PLC., Cambridge, UK), PFKP (cat. no. sc-514824; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), PFK-1 (cat. no. sc-166722; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (cat. no. HRP-60008; 1:5000 dilution; ProteinTech Group, Inc., Chicago, IL, USA).

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: Journal of translational medicine

Article Title: Ubiquitin-specific protease 14 targets PFKL-mediated glycolysis to promote the proliferation and migration of oral squamous cell carcinoma.

doi: 10.1186/s12967-024-04943-z

Figure Lengend Snippet: Fig. 3 USP14 interacts with PFKL. A The exogenous expression of HA-USP14 in the pull-down elution was validated by Western blotting. B Representative images of silver-stained protein bands for mass spectrometry (MS) analysis of USP14-interacting proteins. C Venn diagram showed the numbers of mouse IgG and HA antibody binding proteins identified by immunoprecipitation coupled with mass spectrometry analysis (IP-MS). D Co-immunoprecipitation (Co-IP) analysis of the interactions between exogenous USP14 and endogenous PFKL. Whole-cell lysates from HN4 cells stably expressing HA-USP14 were immunoprecipitated and immunoblotted with antibodies against the indicated proteins. E Co-IP analysis of the interactions between endogenous USP14 and PFKL in HN4 cells. F, G Co-IP analysis of the interactions between exogenous USP14 and PFKL in HEK293T cells. Whole-cell lysates from HEK293T cells stably expressing HA-USP14 and Flag-PFKL were immunoprecipitated and immunoblotted with antibodies against the indicated proteins. H Confocal microscopic analysis of USP14 and PFKL subcellular localization. HN6 cells were fixed and immunoblotted with antibodies against the indicated proteins. Representative images from biological triplicate experiments are shown. Scale bar, 10 μM. I Nuclear and cytoplasmic proteins in three OSCC cell lines were separated by protein extraction kit and immunoblotted with antibodies against the indicated proteins

Article Snippet: Primary antibodies against the following proteins were used for Western blotting analysis: USP14 (cat. no. sc-398009; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Flag (cat. no. 20543-1-AP; 1:2000 dilution; ProteinTech Group, Inc., Chicago, Illinois, USA), HA (cat. no. #2367; 1:1000 dilution; Cell Signaling Technology, Inc., Boston, MA, USA), Ub (cat. no. #3936; 1:1000 dilution; Cell Signaling Technology, Inc., Boston, MA, USA), PFKL (cat. no. ab97443; 1:2000 dilution; Abcam, PLC., Cambridge, UK), PFKP (cat. no. sc-514824; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), PFK-1 (cat. no. sc-166722; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (cat. no. HRP-60008; 1:5000 dilution; ProteinTech Group, Inc., Chicago, IL, USA).

Techniques: Expressing, Western Blot, Staining, Mass Spectrometry, Binding Assay, Immunoprecipitation, Protein-Protein interactions, Co-Immunoprecipitation Assay, Stable Transfection, Protein Extraction

Journal: Journal of translational medicine

Article Title: Ubiquitin-specific protease 14 targets PFKL-mediated glycolysis to promote the proliferation and migration of oral squamous cell carcinoma.

doi: 10.1186/s12967-024-04943-z

Figure Lengend Snippet: Fig. 7 Oncogenic properties of USP14 in oral squamous cell carcinoma xenograft mouse models. A–C A total of 2 × 106 HN6 cells stably transfecting with either shCtrl, shUSP14-1#, or shUSP14-1# + PFKL were subcutaneously injected into nude mice to establish an OSCC xenograft mouse model. After 3 weeks, tumors from 15 of the mice were extracted and photographed. Tumor images (A), tumor volume (B) and weight (C) were then assessed. D Expression pattern of Ki-67 was examined by immunohistochemistry analysis in the xenograft tumors of each group. Original magnification, ×100. **P < 0.01, ***P < 0.001

Article Snippet: Primary antibodies against the following proteins were used for Western blotting analysis: USP14 (cat. no. sc-398009; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Flag (cat. no. 20543-1-AP; 1:2000 dilution; ProteinTech Group, Inc., Chicago, Illinois, USA), HA (cat. no. #2367; 1:1000 dilution; Cell Signaling Technology, Inc., Boston, MA, USA), Ub (cat. no. #3936; 1:1000 dilution; Cell Signaling Technology, Inc., Boston, MA, USA), PFKL (cat. no. ab97443; 1:2000 dilution; Abcam, PLC., Cambridge, UK), PFKP (cat. no. sc-514824; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), PFK-1 (cat. no. sc-166722; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (cat. no. HRP-60008; 1:5000 dilution; ProteinTech Group, Inc., Chicago, IL, USA).

Techniques: Stable Transfection, Injection, Expressing, Immunohistochemistry

Journal: Journal of translational medicine

Article Title: Ubiquitin-specific protease 14 targets PFKL-mediated glycolysis to promote the proliferation and migration of oral squamous cell carcinoma.

doi: 10.1186/s12967-024-04943-z

Figure Lengend Snippet: Fig. 8 Diagram illustrating the USP14-mediated deubiquitylation and stabilization of PFKL

Article Snippet: Primary antibodies against the following proteins were used for Western blotting analysis: USP14 (cat. no. sc-398009; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Flag (cat. no. 20543-1-AP; 1:2000 dilution; ProteinTech Group, Inc., Chicago, Illinois, USA), HA (cat. no. #2367; 1:1000 dilution; Cell Signaling Technology, Inc., Boston, MA, USA), Ub (cat. no. #3936; 1:1000 dilution; Cell Signaling Technology, Inc., Boston, MA, USA), PFKL (cat. no. ab97443; 1:2000 dilution; Abcam, PLC., Cambridge, UK), PFKP (cat. no. sc-514824; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), PFK-1 (cat. no. sc-166722; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (cat. no. HRP-60008; 1:5000 dilution; ProteinTech Group, Inc., Chicago, IL, USA).

Techniques:

Journal: Cell Death & Disease

Article Title: Proteasome-associated deubiquitinase ubiquitin-specific protease 14 regulates prostate cancer proliferation by deubiquitinating and stabilizing androgen receptor

doi: 10.1038/cddis.2016.477

Figure Lengend Snippet: USP14 inhibition or silence reduced cell proliferation in androgen-responsive cancer cells. ( a ) Total proteins were extracted from the cultured cells and subjected to western blot analysis using antibodies against USP14 and AR. GAPDH was used as a loading control. ( b ) LNcap cells were treated with the indicated concentrations of IU1 for 24, 48 and 72 h. ( c ) LNcap cells were treated with siRNA for 24, 48 and 72 h. ( d ) LNcap cells were treated with shRNA for 24, 48 and 72 h. Cell viability was detected by MTS assay. Error bars correspond to 95% confidence intervals of three independent experiments. * P <0.05, # P <0.01 using two-sided t -test. ( e ) LNcap cells exposed to IU1 50 μ M or shRNA 48 h were suspended in 30% agarose for 2 weeks, representative images were shown, and ( f) the numbers of colonies were counted. Error bars correspond to 95% confidence intervals. # P <0.01, compared with control treatments. ( g ) DU145 or PC3 cells were treated with the indicated concentrations of IU1 for 48 h. ( h ) DU145 or PC3 cells were treated with siRNA for 48 h. Cell viability was detected with MTS assay. Error bars correspond to 95% confidence intervals of three independent experiments

Article Snippet: To knock down USP14 expression in prostate cancer cells, siRNA or shRNA targeting human USP14 were synthesized and purchased from Santa Cruz Biotechnology Inc.. siRNA or shRNA with non-specific sequences were used as control scrambled RNA.

Techniques: Inhibition, Cell Culture, Western Blot, Control, shRNA, MTS Assay

Journal: Cell Death & Disease

Article Title: Proteasome-associated deubiquitinase ubiquitin-specific protease 14 regulates prostate cancer proliferation by deubiquitinating and stabilizing androgen receptor

doi: 10.1038/cddis.2016.477

Figure Lengend Snippet: USP14 inhibition or silence failed to induce cell death in LNcap cells. LNcap cells were treated with the indicated concentrations of IU1 or USP14 siRNA for 48 h. The cultured cells were collected and stained with Annexin V-FITC/ PI, followed by flow cytometry analysis. The representative images ( a, c ) and summary of cell death ( b, d ) were shown. Mean±S.D. ( n =3). DM, DMSO. ( e ) LNcap cells were treated with the indicated concentrations of IU1 or USP14 siRNA for 48 h. Total proteins were extracted and subjected to western blot analyses for PARP, p53 or Bax. GAPDH was used as a loading control

Article Snippet: To knock down USP14 expression in prostate cancer cells, siRNA or shRNA targeting human USP14 were synthesized and purchased from Santa Cruz Biotechnology Inc.. siRNA or shRNA with non-specific sequences were used as control scrambled RNA.

Techniques: Inhibition, Cell Culture, Staining, Flow Cytometry, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Proteasome-associated deubiquitinase ubiquitin-specific protease 14 regulates prostate cancer proliferation by deubiquitinating and stabilizing androgen receptor

doi: 10.1038/cddis.2016.477

Figure Lengend Snippet: USP14 inhibition or silence induced cell cycle arrest in LNcap cells. ( a, c ) Shown are representative histograms of PI staining of LNcap. Fluorescence activated cell sorting analysis was performed on LNcap cells that stably expressed USP14 shRNA or control shRNA, or were exposed to the indicated concentrations of IU1 or USP14 siRNA for 24, 48 and 72 h. Three independent experiments were performed. The percentage of cells in each population in each cell cycle phase at 48 h was calculated ( b, d, e )

Article Snippet: To knock down USP14 expression in prostate cancer cells, siRNA or shRNA targeting human USP14 were synthesized and purchased from Santa Cruz Biotechnology Inc.. siRNA or shRNA with non-specific sequences were used as control scrambled RNA.

Techniques: Inhibition, Staining, Fluorescence, FACS, Stable Transfection, shRNA, Control

Journal: Cell Death & Disease

Article Title: Proteasome-associated deubiquitinase ubiquitin-specific protease 14 regulates prostate cancer proliferation by deubiquitinating and stabilizing androgen receptor

doi: 10.1038/cddis.2016.477

Figure Lengend Snippet: USP14 regulates G1-S transition. ( a − e ) Representative images of western blot analyses for key proteins associated with G1-S phase transition. Total proteins were extracted from LNcap cells that had been manipulated as described below, and subjected to western blot analyses for CDK6, CDK4, CDK2, cyclinD1, p27, p15, phospho-Rb (p-Rb) and Rb. The LNcap cells were treated with the indicated concentrations of IU1 ( a ), with 50 μ M IU1 for the indicated durations ( b ), with transfection of USP14 siRNA or control siRNA for 48 h ( c ), with stable expression of USP14 shRNA or control shRNA ( d ), or with transfection with Flag-USP14 plasmids or control vectors ( e ). GAPDH was used as a loading control. Three independent repeats were performed for each experiment. ( f ) MTS assay for LNcap cells transfected with Flag-USP14 plasmids or control vectors for 24, 48 and 72 h. Error bars correspond to 95% confidence intervals of three independent experiments. * P <0.05 using two-sided t -test

Article Snippet: To knock down USP14 expression in prostate cancer cells, siRNA or shRNA targeting human USP14 were synthesized and purchased from Santa Cruz Biotechnology Inc.. siRNA or shRNA with non-specific sequences were used as control scrambled RNA.

Techniques: Western Blot, Sublimation, Transfection, Control, Expressing, shRNA, MTS Assay

Journal: Cell Death & Disease

Article Title: Proteasome-associated deubiquitinase ubiquitin-specific protease 14 regulates prostate cancer proliferation by deubiquitinating and stabilizing androgen receptor

doi: 10.1038/cddis.2016.477

Figure Lengend Snippet: USP14 inhibits the expression and phosphorylation of MDM2, stabilizes AR, and increases PSA level. ( a-d ) Representative images of western blot analyses for AR, PSA, MDM2 and Ser166-phosphorylated MDM2 (p-MDM2) in the total proteins extracted from LNcap cells that were treated with the indicated concentrations of IU1 ( a ), IU1 50 μ M for the indicated duration ( b ), or transfection of USP14 siRNA or control siRNA (CTR) ( c ), or from LNcap cells stably expressing USP14 shRNA or control shRNA ( d ). GAPDH was used as a loading control. Three independent experiments were performed. ( e ) LNcap cells stably expressing Flag-USP14 or control vector were harvested. Total proteins were extracted and subjected to western blot analyses for AR, PSA, MDM2 and p-MDM2. GAPDH was used as a loading control. Flag and GFP were used as indicators of transfection efficiency. ( f ) LNcap cells were exposed to the indicated concentrations of IU1 in the absence or presence of with proteasome inhibitor bortezomib (Vel, 50 nM) for 48 h. Total proteins were extracted and subjected to western blot analyses for AR

Article Snippet: To knock down USP14 expression in prostate cancer cells, siRNA or shRNA targeting human USP14 were synthesized and purchased from Santa Cruz Biotechnology Inc.. siRNA or shRNA with non-specific sequences were used as control scrambled RNA.

Techniques: Expressing, Phospho-proteomics, Western Blot, Transfection, Control, Stable Transfection, shRNA, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Proteasome-associated deubiquitinase ubiquitin-specific protease 14 regulates prostate cancer proliferation by deubiquitinating and stabilizing androgen receptor

doi: 10.1038/cddis.2016.477

Figure Lengend Snippet: USP14 reduces the ubiquitination of AR and stabilizes AR proteins. ( a ) LNcap cells were exposed to IU1 50 μ M or USP14 siRNA for 24 h. Total RNAs were extracted and subjected to RT 2 -PCR analysis. GAPDH was used as an internal control. Three independent experiments were performed. Mean±S.D. ( n =3). * P <0.05, compared with DM. # P <0.01, compared with scramble siRNA. ( b ) LNcap cells were exposed to IU1 50 μ M or USP14 siRNA for 48 h, immunoprecipitated with AR antibody beads and immunoblotted for ubiquitin (Ub) and AR. Cells were treated with MG132 (10 μ M) for 6 h before harvest. ( c ) LNcap cells were exposed to IU1 50 μ M or USP14 siRNA for 48 h, immunoprecipitated with MDM2 antibody beads and immunoblotted for AR and MDM2. ( d ) Total proteins were extracted from LNcap cells, immunoprecipitated with USP14 antibody beads and immunoblotted for AR and USP14 (upper), and immunoprecipitated with AR antibody beads and immunoblotted for AR and USP14 (lower)

Article Snippet: To knock down USP14 expression in prostate cancer cells, siRNA or shRNA targeting human USP14 were synthesized and purchased from Santa Cruz Biotechnology Inc.. siRNA or shRNA with non-specific sequences were used as control scrambled RNA.

Techniques: Ubiquitin Proteomics, Control, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Proteasome-associated deubiquitinase ubiquitin-specific protease 14 regulates prostate cancer proliferation by deubiquitinating and stabilizing androgen receptor

doi: 10.1038/cddis.2016.477

Figure Lengend Snippet: A molecular model for USP14 to regulate AR and cell cycle. MDM2 is one of the E3 ligases for AR that attaches ubiquitin (Ub) chain to AR and thereby leads to AR degradation. On one hand, USP14 indirectly decreases the ubiquitination and degradation of AR by decreasing the MDM2 protein level; on the other hand, USP14 directly deubiquitinates AR by binding AR and removing the ubiquitin chain from AR and thereby antagonizes ubiquitin proteasome system-mediated AR degradation. AR mediates cell growth and proliferation by transcriptional activation of PSA expression and promoting G1-S phase transition

Article Snippet: To knock down USP14 expression in prostate cancer cells, siRNA or shRNA targeting human USP14 were synthesized and purchased from Santa Cruz Biotechnology Inc.. siRNA or shRNA with non-specific sequences were used as control scrambled RNA.

Techniques: Ubiquitin Proteomics, Binding Assay, Activation Assay, Expressing, Sublimation

Journal: Cancer Research

Article Title: Proteasome Function Is Required for DNA Damage Response and Fanconi Anemia Pathway Activation

doi: 10.1158/0008-5472.can-07-1015

Figure Lengend Snippet: Figure 2. Depletion of proteasome subunits inhibits the activation of the Fanconi anemia pathway. (Full-length blots/gels are presented in Supplementary Fig. S3E and F). A, efficiency of proteasome subunit depletion. Lanes 1 to 8, semiquantitative RT-PCR analysis of mRNA expression in PSMB3-, PSMD4-, DSS1-, PSMD14-, USP14-, and UCH37-depleted cells 48 h after siRNA transfection (depletion efficiency was 75% for PSMB3, 65% for PSMD4, 44% for DSS1, 63% for PSMD14, 45% for USP14, and 43% for UCH37); lanes 9 to 16, whole-cellular extracts were also subjected to PSMB3, PSMD4, and USP14 immunoblots (depletion efficiency was 86% for PSMB3, 99% for PSMD4, and 79% for USP14). PSMD14 depletion led to a concomitant down-regulation of PSMD4 protein expression probably due to the destabilization of the 19S proteasome induced by the lack of PSMD14 protein. B, depletion of PSMD14 strongly inhibited proteasome function, whereas depletion of PSMB3 and PSMD4 had a mild effect and depletion of USP14, UCH37, and DSS1 had only a minor effect on proteasome function. GFPu-1 cells expressing GFP fused to a short degron were used to assess proteasome proteolytic function. Forty-eight hours after siRNA transfection, as well as 6 h after treatment with MG132 (2 Amol/L) and bortezomib (1 Amol/L), cells were harvested, washed in PBS, and analyzed by flow cytometry for GFP expression. Representative flow cytometry histograms. The experiments were independently repeated thrice with consistent results. C, IR-induced FANCD2 monoubiquitination was strongly inhibited by PSMD14 depletion, minimally affected by PSMB3, PSMD4, DSS1, USP14, and UCH37 depletion. Forty hours after siRNA transfection, HeLa cells were irradiated and treated with/without MG132 for 8 h after IR. Whole-cell extracts were subjected to FANCD2 immunoblotting. D, depletion of PSMB3, PSMD4, PSMD14, and USP14, but not DSS1 and UCH37, led to inhibition of IR-induced FANCD2 foci formation. Cells were treated as in (B). Representative pictures of cells immunostained with anti-FANCD2 antibody. Percentage of cells harboring at least five FANCD2 foci before (white columns), 8 h after IR (gray columns), and 8 h after IR in the presence of MG132 (black columns). Bars, SE. *, significant difference with irradiated control siRNA–transfected cells (P < 0.05, unpaired t test). Bar, 20 Am.

Article Snippet: Mouse monoclonal antibodies directed against FANCD2 (FI-17, 1:200 dilution; Santa Cruz Biotechnology), gH2AX (#05-636, 1:1,000; Upstate), proliferating cell nuclear antigen (PCNA; PC10, 1:1,000; Santa Cruz Biotechnology), PSMB3 (PW8130, 1:1,000; Biomol International), PSMD4 (PW9250, 1:1,000; Biomol International), USP14 (H00009097-M04, 1:2,000; Novus Biologicals), and rabbit polyclonal antibodies against USP1 [C-term, 1:3,000; gift of Drs. Tony Huang (New York University School of Medicine, New York, NY) and Alan D’Andrea (Dana Farber Cancer Institute, Boston, MA); ref. 24] were used as primary antibodies.

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Western Blot, Flow Cytometry, Irradiation, Inhibition, Control

Journal: Cancer Research

Article Title: Proteasome Function Is Required for DNA Damage Response and Fanconi Anemia Pathway Activation

doi: 10.1158/0008-5472.can-07-1015

Figure Lengend Snippet: Figure 5. Depletion of PSMB3, PSMD4, PSMD14, and USP14 inhibits IR-induced foci formation of BRCA1 and RAD51, whereas depletion of DSS1 inhibits only RAD51 foci formation. Forty hours after siRNA transfection, HeLa cells were irradiated (15 Gy) and fixed 8 h after IR. Cells were double stained with anti-BRCA1 and anti-RAD51 antibodies. Representative pictures of immunostained cells are shown together with quantification of the cells with at least five BRCA1 or RAD51 foci before (; white columns) and 8 h after IR (+; gray columns). Columns, mean of four independent experiments; bars, SE. *, significant difference with irradiated control siRNA–transfected cells (P < 0.05, unpaired t test). Bar, 20 Am.

Article Snippet: Mouse monoclonal antibodies directed against FANCD2 (FI-17, 1:200 dilution; Santa Cruz Biotechnology), gH2AX (#05-636, 1:1,000; Upstate), proliferating cell nuclear antigen (PCNA; PC10, 1:1,000; Santa Cruz Biotechnology), PSMB3 (PW8130, 1:1,000; Biomol International), PSMD4 (PW9250, 1:1,000; Biomol International), USP14 (H00009097-M04, 1:2,000; Novus Biologicals), and rabbit polyclonal antibodies against USP1 [C-term, 1:3,000; gift of Drs. Tony Huang (New York University School of Medicine, New York, NY) and Alan D’Andrea (Dana Farber Cancer Institute, Boston, MA); ref. 24] were used as primary antibodies.

Techniques: Transfection, Irradiation, Staining, Control

Journal: Nature

Article Title: USP14-regulated allostery of the human proteasome by time-resolved cryo-EM.

doi: 10.1038/s41586-022-04671-8

Figure Lengend Snippet: Fig. 1 | Time-resolved cryo-EM analysis of the conformational landscape of USP14–proteasome complexes in the act of substrate degradation. a, b, Cryo-EM density map of the substrate-engaged USP14–proteasome complex in state ED2.1 USP14, viewed from the top (a) and side (b). c, Side view of the cryo-EM density map of the substrate-engaged USP14–proteasome complex in state ED4 USP14. Compared to the view of ED2.1 USP14 in b, USP14 is rotated about 30° to dock onto the AAA domain of RPT1. To visualize the substrate density inside the AAA-ATPase motor, the density of RPT5 is omitted in both b and c. d, Atomic model of state ED2.1 USP14 viewed from the same perspective as in a. e, Kinetic changes of overall particle populations of SD-like and ED-like states versus EA-like states obtained from time-resolved cryo-EM analysis. EA-like states include EA1 UBL, EA2.0 UBL and EA2.1 UBL. SD-like states include SB USP14, SC USP14 SD4 USP14 and SD5 USP14.

Article Snippet: Western blot was used to detect RPN13 and USP14 in the proteasomes using anti-RPN13 antibody (Abcam, 1:10,000 dilution) and anti-USP14 antibody (Novus, 1:1,000 dilution).

Techniques: Cryo-EM Sample Prep

Journal: Nature

Article Title: USP14-regulated allostery of the human proteasome by time-resolved cryo-EM.

doi: 10.1038/s41586-022-04671-8

Figure Lengend Snippet: Fig. 2 | Structural basis of proteasome-mediated activation of USP14. a, Side-chain interactions between the USP14 UBL domain and the RPN1 T2 site in the proteasome state ED2.1 USP14. b, Structural comparison of the blocking loops by superimposing the USP14 structure in state ED2.1 USP14 with two crystal structures of USP14 in its isolated form (PDB ID: 2AYN) and in complex with ubiquitin aldehyde5 (UbAl) (PDB ID: 2AYO). c, Magnified view of the ubiquitin–USP–OB sandwich architecture in state ED2.1 USP14. d, Local cryo-EM density of the BL1 motif in state ED2.1 USP14 in mesh representation superimposed with its atomic model in cartoon representation from two opposite orientations, showing its β-hairpin conformation. e–g, Magnified views of the interfaces between the catalytic Cys114 of USP14 and the C-terminal Gly76 of ubiquitin (e), between the USP14 BL1 motif and the RPT1 OB domain (f), and between the USP14 BL3 motif and the RPT2 OB domain (g). Key residues mediating the inter-molecular interactions are shown in stick representation in a–g. h, In vitro degradation of

Article Snippet: Western blot was used to detect RPN13 and USP14 in the proteasomes using anti-RPN13 antibody (Abcam, 1:10,000 dilution) and anti-USP14 antibody (Novus, 1:1,000 dilution).

Techniques: Activation Assay, Comparison, Blocking Assay, Isolation, Ubiquitin Proteomics, Cryo-EM Sample Prep, In Vitro

Journal: Nature

Article Title: USP14-regulated allostery of the human proteasome by time-resolved cryo-EM.

doi: 10.1038/s41586-022-04671-8

Figure Lengend Snippet: Fig. 3 | Structural dynamics and mechanism of allosteric regulation of the AAA-ATPase motor by USP14. a, b, Side-by-side comparison of the USP14– ATPase subcomplex structures aligned against the CP in six substrate-engaged states (a) and four substrate-inhibited states (b). c, d, Plots of distance from the pore-1 loop of each ATPase to the CP (c) and to the substrate (d) in distinct states. e, The AAA domain structures of the ATPase motor in six substrate-engaged states. f, Varying architecture of the pore-1 loop staircase interacting with the substrate in distinct states. The distances from disengaged pore-1 loops to the substrate are marked. The side chains of the pore-1 loop residues, featuring a consensus sequence of K/M-Y/F-V/L/I, are shown in stick representation, with the aromatic residues highlighted in transparent sphere representation. g, Electrostatic surface representation of the full-length USP14, coloured according to electrostatic potential from red (−5.0 kT e−1,

Article Snippet: Western blot was used to detect RPN13 and USP14 in the proteasomes using anti-RPN13 antibody (Abcam, 1:10,000 dilution) and anti-USP14 antibody (Novus, 1:1,000 dilution).

Techniques: Comparison, Sequencing

Journal: Nature Communications

Article Title: Design of quinoline SARS-CoV-2 papain-like protease inhibitors as oral antiviral drug candidates

doi: 10.1038/s41467-025-56902-x

Figure Lengend Snippet: a Differential scanning fluorimetry assay of Jun13296 in stabilizing SARS-CoV-2 PL pro . Jun12682 was included as a positive control for comparison. Data from Jun12682 is the mean of two repeats, and data from Jun13296 is the mean ± standard deviation of three technical repeats. b K i plot of Jun13296 in inhibiting SARS-CoV-2 PL pro hydrolysis of ISG15-AMC. c K i plot of Jun13296 in inhibiting SARS-CoV-2 PL pro hydrolysis of Ub-AMC. d Counter screening of Jun 13296 against host proteases USP2, USP7, USP8, USP14, USP15, USP30, UCH-L1, cathepsin B, cathepsin K, calpain-1, trypsin, and caspase 3. Data in ( d ) are presented as mean ± standard deviation of two technical repeats. Source data are provided as a file.

Article Snippet: E-520-025), 500 nM USP14 (ProSci, 91-171), 1 nM USP15 (R&D systems, E594), 20 nM USP30 (R&D systems, E582), and 1 nM UCH-L1 (R&D systems, 6007-CY).

Techniques: Fluorimetry Assay, Positive Control, Comparison, Standard Deviation

Journal: Cell reports

Article Title: Proteasome activity maintains cell-type-specific gene expression

doi: 10.1016/j.celrep.2026.116973

Figure Lengend Snippet: (A) Lysates from HCT116 WT, ΔRAZUL, or hRpn10 VWA cells were resolved and analyzed by immunoprobing for hRpn10, E6AP, or β-actin. (B) Lysates from WT, ΔhRpn13, trRpn13, ΔRAZUL, hRpn10 VWA , or ΔUCHL5 cells were resolved and immunoprobed for K48-linked (left) or bulk (right) ubiquitin chains or β-actin. (C) Immunoblots probing for ubiquitin or hRpn2 of proteasomes immunoprecipitated by anti-hRpt3 antibodies from WT, ΔhRpn13, trRpn13, ΔRAZUL, hRpn10 VWA , or ΔUCHL5 without (left) or with (right) DSP crosslinker. IgG antibodies were included as a control. (D) Boxplot representation plotting the range and mean value of protein abundance for hRpn1, hRpn10, hRpn8, hRpn11, hRpn13, hRpn2, hRpt3, β5, E6AP, UCHL5, and USP14 across three samples as measured by TMT-MS. (E) Lysates from HCT116 WT, ΔhRpn13, trRpn13, ΔRAZUL, hRpn10 VWA , or ΔUCHL5 cells resolved and analyzed by immunoprobing for hRpn1, hRpn2, hRpn10, hRpn13, hRpn8, hRpn11, USP14, UCHL5, β5, or β-actin. (F) Graphical plot of protein and mRNA abundance in hRpn10 VWA compared to WT by TMT-MS ( x axis) and qPCR ( y axis), respectively, for RP subunits hRpn1, hRpn10, hRpn11, and hRpn13, CP β5, or deubiquitinases UCHL5 and USP14. A dotted line indicates no change. (G) Volcano plot of fold-change in protein abundance ( x axis, log 2 ) and adjusted p value ( y axis, −log 10 ) in hRpn10 VWA cells relative to WT. Vertical and horizontal lines are included at ±1 and 1.3, respectively. Proteins with expression values statistically greater or less than 2-fold difference are colored and labeled according to their molecular function as indicated in the legend. (H) Plot of the change in protein abundance (log 2 ) relative to WT by TMT-MS for ΔRAZUL ( x axis) and hRpn10 VWA ( y axis). The Spearman rank correlation value is included. Data with adjusted p value ≥ 0.05 are excluded from this analysis. Color coding follows (G). FC, fold change. (I) Illustration of the aftereffects that arise following deletion of hRpn10 UIMs and RAZUL domain. Weak binding of ubiquitinated substrates to proteasomes causes dysregulated protein levels in multiple functional pathways and induces upregulated gene expression of proteasome subunits. hRpn10 VWA , ubiquitin, substrate, peptide, RP, and CP α or β subunits are colored purple, yellow, brown, dark gray, light yellow, gray, or light gray, respectively. See also , , and .

Article Snippet: TaqMan probe for USP14 , Applied Biosystem , Hs00193036_m1.

Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Control, Quantitative Proteomics, Expressing, Labeling, Binding Assay, Functional Assay, Gene Expression