Journal: Molecular and Cellular Biology

Article Title: Impact of Losing hRpn13 Pru or UCHL5 on Proteasome Clearance of Ubiquitinated Proteins and RA190 Cytotoxicity

doi: 10.1128/MCB.00122-20

Figure Lengend Snippet: Generation of a cell line expressing truncated hRpn13 (trRpn13) competent for binding UCHL5 but not proteasome. (A) Schematic representation of the hRpn13-expressing ADRM1 gene highlighting and labeling each forward strand exon, including noncoding exon 1 and gRNA-targeted exon 2. Exons 3 to 10, as well as the ATG codon in exon 3 encoding M109, are also indicated. (B) Structure of hRpn13 (PDB 2KR0) highlighting exons of the ADRM1 gene colored as displayed in panel A. Exons 1 to 4 and 8 to 10 express the hRpn13 Pru and DEUBAD domains, respectively, with exon 7 yielding a helix that bridges these two structural domains. Exons 5 and 6 express parts of the protein that are intrinsically disordered and are omitted from this figure. The side chain heavy atoms are displayed (pink) for M109, which is located at the end of a helix encoded by exon 3. (C, top) Whole-cell extract from HCT116 (WT) or trRpn13 cells was resolved and analyzed by immunoprobing for hRpn13, hRpn2, or hRpt3, as indicated, with β-actin used as a loading control. (Bottom) Proteasomes from WT or trRpn13 whole-cell extract were immunoprecipitated (IP) with anti-Rpt3 antibodies and immunoprobed for hRpn13 or hRpn2 as a positive control. (D) Total RNA from HCT116 (WT) or trRpn13 was reverse transcribed to cDNA and subjected to PCR for evaluation with primers targeting the indicated ADRM1 exon junctions. PCR products were run on a 1% agarose gel and visualized by SYBR safe DNA gel stain. (E) Sashimi plot depicting normalized coverage for the ADRM1 gene that expresses hRpn13 in HCT116 trRpn13 or WT cells. (Top) Counts-per-minute (CPM)-normalized expression is shown along the y axis for the length of the gene along the x axis. Reads spanning splice junctions are depicted as arcs annotated with CPM-normalized counts. (Bottom) Schematic of the primary transcript (ENST00000253003) for the gene from the Ensembl database, version 75, with exons shown as boxes, introns shown as lines, and arrows indicating the direction of transcription. Numbers at the bottom denote the chromosomal coordinates along chromosome 20. (F) Sanger sequencing analysis of hRpn13 cDNA from WT or trRpn13 cells denoting the location of the two sgRNAs (red arrows), 5′ UTR, which includes exon 1 (gray arrow), and protein-coding exon 2 and exon 3 (yellow bars). An expansion is included in the lower panel showing the 5′ and 3′ portions from the deletion of exon 2. This image was generated by using Geneious. (G) Lysates from WT, ΔhRpn13, or trRpn13 cells were immunoprobed for UCHL5, hRpn13, or β-actin (as a loading control). (H) Lysates from WT, ΔhRpn13, or trRpn13 cells treated for 30 min with the cross-linker DSP were subjected to immunoprecipitation with anti-Rpn13 antibodies, and the immunoprecipitants were immunoprobed for UCHL5 or hRpn13 as indicated. Immunoblots of the whole-cell extract (WCE) are included as indicated in the lower panels for UCHL5, hRpn13, or β-actin (as a loading control).

Article Snippet: UCHL5 and β-actin mRNA expression was measured on a quantitative RT-PCR (CFX384; Bio-Rad) instrument using TaqMan probes for UCHL5 (Hs01044470_m1; Thermo Fisher Scientific) and β-actin (Hs01060665_g1; Thermo Fisher Scientific). β-Actin was used as an internal standard.

Techniques: Expressing, Binding Assay, Labeling, Control, Immunoprecipitation, Positive Control, Reverse Transcription, Agarose Gel Electrophoresis, Staining, Sequencing, Generated, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Impact of Losing hRpn13 Pru or UCHL5 on Proteasome Clearance of Ubiquitinated Proteins and RA190 Cytotoxicity

doi: 10.1128/MCB.00122-20

Figure Lengend Snippet: Levels of ubiquitinated proteins at the proteasome are altered in cells deleted of hRpn13, UCHL5, or the hRpn13 Pru. (A) Schematic representation of the UCHL5 gene from chromosome 1 depicting and labeling the exons as well as the gRNA targeting of exon 1 to generate the ΔUCHL5 cell line. (B) Sashimi plots depicting normalized coverage for the UCHL5 gene in HCT116 ΔUCHL5 or WT cells. (Top) CPM-normalized expression is shown along the y axis for the length of the gene along the x axis. Reads spanning splice junctions are depicted as arcs annotated with CPM-normalized counts. (Bottom) Schematic of the primary transcript (ENST00000367455) for the gene from the Ensembl database, version 75, with exons shown as boxes, introns shown as lines, and arrows indicating the direction of transcription. Numbers at the bottom denote the chromosomal coordinates along chromosome 1. (C) Total RNA from WT or ΔUCHL5 cells was reverse transcribed to cDNA and subjected to TaqMan PCR for UCHL5 mRNA analysis. β-Actin was used as an internal standard, and the data were normalized to the WT by using the 2–ΔΔCT method. Reported values represent means, with error bars indicating standard errors of the means (SEM) for n = 6. Fold change is also indicated for ΔUCHL5 compared to the WT. ****, P < 0.0001 by Student's t test analysis. (D) Lysates from WT or ΔUCHL5 cells were resolved and analyzed by immunoprobing for hRpn13, hRpt3, or hRpn2, as indicated, with β-actin as a loading control. (E) Whole-cell extract (WCE) from WT, ΔhRpn13, trRpn13, or ΔUCHL5 cells were resolved and analyzed by immunoprobing for ubiquitin (Ub), hRpn13, UCHL5, or proteasome components hRpn1, hRpn2, hRpn10, hRpn11, hRpt3, or USP14, as indicated. β-Actin was used as a loading control. Graphical plots show protein levels in ΔhRpn13, trRpn13, or ΔUCHL5 cells relative to the WT after normalization to β-actin for ubiquitin (Ub) levels in the region bracketed (left), hRpn1, hRpn2, hRpn10, hRpn11, hRpt3, and USP14. Data are plotted as average fold changes ± SEM for three independent experiments. (F) Proteasomes from WT, ΔhRpn13, trRpn13, or ΔUCHL5 cells were immunoprecipitated (IP) with anti-Rpt3 antibodies and the immunoprecipitates immunoprobed for ubiquitin (Ub), hRpn13, UCHL5, or proteasome components hRpn1, hRpn2, hRpn10, hRpn11, or hRpt3, as indicated. Graphical plots indicate protein levels in ΔhRpn13, trRpn13, or ΔUCHL5 cells relative to the WT after normalization to hRpn2 for ubiquitin (Ub) levels in the region bracketed (left), hRpn1 and hRpn10. Data are plotted as average fold changes ± SEM for three independent experiments. Bulk ubiquitin was probed with antiubiquitin/P4D1 (3936; Cell Signaling Technology). Dashed lines are included for the plots in panels E and F at a value of 1.0.

Article Snippet: UCHL5 and β-actin mRNA expression was measured on a quantitative RT-PCR (CFX384; Bio-Rad) instrument using TaqMan probes for UCHL5 (Hs01044470_m1; Thermo Fisher Scientific) and β-actin (Hs01060665_g1; Thermo Fisher Scientific). β-Actin was used as an internal standard.

Techniques: Labeling, Expressing, Reverse Transcription, Control, Ubiquitin Proteomics, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: Impact of Losing hRpn13 Pru or UCHL5 on Proteasome Clearance of Ubiquitinated Proteins and RA190 Cytotoxicity

doi: 10.1128/MCB.00122-20

Figure Lengend Snippet: Transient loss of hRpn13 or UCHL5 disrupts cell cycle progression in HCT116 cells. (A) Metabolic activity of WT, ΔhRpn13, trRpn13, or ΔUCHL5 cells measured by MTT assay at 48 h postseeding for the indicated cell density (n = 6). (B) Representative image (top) and plot (bottom) for flow cytometry analyses of WT, ΔhRpn13, trRpn13, or ΔUCHL5 cells from three independent experiments with triplicate repetitions. The cells were seeded at 0.4 million cells per well in 6-well plates for 48 h and subsequently labeled with EdU and propidium iodide. The distribution of cells in G0/G1, S, and G2/M is shown (bottom) by plotting the mean and SEM (error bar) for each cell line. (C) WT cells treated with scrambled control RNA or siRNA targeting hRpn13 (sihRpn13) or UCHL5 (siUCHL5) for 48 h, followed by labeling with EdU and propidium iodide, were subjected to flow cytometry analyses. A representative image (right) and plot (left) for the distribution of cells in G0/G1, S, and G2/M is provided from three independent experiments with triplicate repetitions. The plot indicates the means and SEM (error bars). ***, P < 0.001; ****, P < 0.0001; two-way ANOVA, Dunnett’s post hoc test. (D, left) Lysates from WT, ΔhRpn13, trRpn13, or ΔUCHL5 cells seeded at 0.4 million cells per well in 6-well plates for 48 h were resolved and immunoprobed for p21Cip1, Wee1, p53, hRpn13, UCHL5, or β-actin (as a loading control) as indicated (representative image). (Right) The quantitation of protein levels normalized to β-actin from three independent experiments is displayed. Averaged values are plotted with error bars indicating SEM. ****, P < 0.0001; analyses were done by Student's t test. (E, left) Lysates from WT cells treated with scrambled control RNA or siRNA targeting hRpn13 (sihRpn13) or UCHL5 (siUCHL5) were resolved and immunoprobed as indicated for p21Cip1, Wee1, p53, hRpn13, UCHL5, or β-actin (as a loading control; shown is a representative image). (Right) Quantitation of protein levels normalized to β-actin across three independent experiments. Averaged values are plotted with error bars indicating SEM (****, P < 0.0001; Student's t test).

Article Snippet: UCHL5 and β-actin mRNA expression was measured on a quantitative RT-PCR (CFX384; Bio-Rad) instrument using TaqMan probes for UCHL5 (Hs01044470_m1; Thermo Fisher Scientific) and β-actin (Hs01060665_g1; Thermo Fisher Scientific). β-Actin was used as an internal standard.

Techniques: Activity Assay, MTT Assay, Flow Cytometry, Labeling, Control, Quantitation Assay

Journal: Molecular and Cellular Biology

Article Title: Impact of Losing hRpn13 Pru or UCHL5 on Proteasome Clearance of Ubiquitinated Proteins and RA190 Cytotoxicity

doi: 10.1128/MCB.00122-20

Figure Lengend Snippet: Loss of hRpn13 Pru, and not UCHL5, attenuates sensitivity at the proteasome to RA190. (A) Ribbon diagram representation of hRpn13 with the missing Pru domain exons (exon 2 and most of exon 3) in yellow, the remaining Pru exon (exon 4) in blue, and the DEUBAD domain and interdomain helix in green. Heavy side chain atoms are displayed for RA190-targeted cysteine 88 (C88, orange) and trRpn13 start site methionine 109 (M109, pink). This image was made by using PDB entry 5IRS. (B and C) Whole-cell extracts (WCE) (B) or immunoprecipitated proteasomes (C) from HCT116 (WT) or trRpn13 cells treated for 24 h with RA190 (1 μM) or DMSO (as a control) were resolved and immunoprobed for ubiquitin (Ub), with β-actin as a loading control (B) or hRpn2 as a positive control (C). The ratio of ubiquitin levels is plotted for the region bracketed from trRpn13 or WT cells normalized first to β-actin (B) or hRpn2 (C) and then to the DMSO control WT. Values are average fold changes ± SEM from three independent experiments. (D and E) WCE (D) or immunoprecipitated proteasomes (E) from WT or ΔUCHL5 cells were resolved and immunoprobed for ubiquitin (Ub), with β-actin as a loading control (D) or hRpt3 as a positive control (E). The ratio of ubiquitin levels is plotted for the region bracketed from ΔUCHL5 or WT cells first normalized to β-actin (D) or hRpt3 (E) and then to the DMSO control WT. Values are average fold changes ± SEM from three independent experiments. Bulk ubiquitin was probed with antiubiquitin antibody (MAB1510; EMD Millipore). Dashed lines are included for the plots in panels B to E at a value of 1.0.

Article Snippet: UCHL5 and β-actin mRNA expression was measured on a quantitative RT-PCR (CFX384; Bio-Rad) instrument using TaqMan probes for UCHL5 (Hs01044470_m1; Thermo Fisher Scientific) and β-actin (Hs01060665_g1; Thermo Fisher Scientific). β-Actin was used as an internal standard.

Techniques: Immunoprecipitation, Control, Ubiquitin Proteomics, Positive Control

Journal: Molecular and Cellular Biology

Article Title: Impact of Losing hRpn13 Pru or UCHL5 on Proteasome Clearance of Ubiquitinated Proteins and RA190 Cytotoxicity

doi: 10.1128/MCB.00122-20

Figure Lengend Snippet: RA190-triggered cell death is reduced in trRpn13 but not UCHL5 cells. (A) Viability of HCT116 (WT), ΔhRpn13, trRpn13, or ΔUCHL5 cells treated for 48 h with the indicated concentration of RA190 or DMSO (as a control), as assessed by MTT assays. (B) WT, ΔhRpn13, trRpn13, or ΔUCHL5 cells were exposed for 24 h to RA190 (1 μM) or DMSO (as a control) and monitored for morphological changes by bright-field microscopy. Arrows indicate blebbing cells undergoing apoptosis. Images shown are representative of two independent experiments (scale bar, 20 μm). (C) WT, ΔhRpn13, trRpn13, or ΔUCHL5 cells treated for 24 h with RA190 (1 μM) or DMSO (as a control) were subjected to flow cytometry analyses after staining with annexin V-FITC and 7-aminoactinomycin D (7-AAD) (representative data are on the left). Population percentage of early and late combined apoptotic or viable cells across three independent experiments is plotted (center) and performed as described for the left. A plot of the ratio of RA190-treated cells to corresponding DMSO control following normalization to the WT is also included for the early apoptotic cell population. The plotted data represent the means and SEM (error bars); *, P < 0.05; ***, P < 0.001; Student's t test. (D) Whole-cell extract from WT, ΔhRpn13, trRpn13, or ΔUCHL5 cells treated for 24 h with RA190 (1 μM), carfilzomib (100 nM), or DMSO (control) was immunoprobed for apoptotic markers caspase 3 (Casp 3) and PARP, hRpn13, UCHL5, or GAPDH (as a loading control; representative image is on the left). Clvd, cleaved. The ratio of cleaved caspase 3 (Clvd-Casp3) to procaspase 3 (Pro-Casp3) or of cleaved PARP (Clvd-PARP) to pro-PARP (Pro-PARP) for RA190- or carfilzomib-treated ΔhRpn13, trRpn13, or ΔUCHL5 cells is plotted normalizing to the respective RA190- or carfilzomib-treated WT cells for three independent experiments, performed as shown on the left. The plotted data represent the means and SEM (error bars). Dashed lines are displayed at a value of 1.0.

Article Snippet: UCHL5 and β-actin mRNA expression was measured on a quantitative RT-PCR (CFX384; Bio-Rad) instrument using TaqMan probes for UCHL5 (Hs01044470_m1; Thermo Fisher Scientific) and β-actin (Hs01060665_g1; Thermo Fisher Scientific). β-Actin was used as an internal standard.

Techniques: Concentration Assay, Control, Microscopy, Flow Cytometry, Staining

Journal: Molecular and Cellular Biology

Article Title: Impact of Losing hRpn13 Pru or UCHL5 on Proteasome Clearance of Ubiquitinated Proteins and RA190 Cytotoxicity

doi: 10.1128/MCB.00122-20

Figure Lengend Snippet: Model summarizing cellular contributions of hRpn13 or UCHL5. Shown is art depicting the impact of hRpn13 Pru domain loss (trRpn13) or UCHL5 loss (ΔUCHL5) on proteasome activity and RA190 targeting. Deletion of the hRpn13 Pru domain (trRpn13) reduces the population of proteasome-bound ubiquitinated proteins (indicated with orange ubiquitin molecules), most likely due to defective recruitment or retention with impact varying depending on substrate ease of unfolding (green or pink represents loosely or more stably folded substrates, respectively). Deletion of UCHL5 (ΔUCHL5) leads to accumulation of ubiquitinated proteins at proteasomes and does not interfere with RA190-triggered cell death.

Article Snippet: UCHL5 and β-actin mRNA expression was measured on a quantitative RT-PCR (CFX384; Bio-Rad) instrument using TaqMan probes for UCHL5 (Hs01044470_m1; Thermo Fisher Scientific) and β-actin (Hs01060665_g1; Thermo Fisher Scientific). β-Actin was used as an internal standard.

Techniques: Activity Assay, Ubiquitin Proteomics, Stable Transfection

Journal: Molecular and Cellular Biology

Article Title: Impact of Losing hRpn13 Pru or UCHL5 on Proteasome Clearance of Ubiquitinated Proteins and RA190 Cytotoxicity

doi: 10.1128/MCB.00122-20

Figure Lengend Snippet: Guide RNAs used for CRISPR/Cas9 gene editing

Article Snippet: UCHL5 and β-actin mRNA expression was measured on a quantitative RT-PCR (CFX384; Bio-Rad) instrument using TaqMan probes for UCHL5 (Hs01044470_m1; Thermo Fisher Scientific) and β-actin (Hs01060665_g1; Thermo Fisher Scientific). β-Actin was used as an internal standard.

Techniques: CRISPR, Sequencing

Journal: BMC Cancer

Article Title: UCHL5 promotes hepatocellular carcinoma progression by promoting glycolysis through activating Wnt/β-catenin pathway

doi: 10.1186/s12885-023-11317-z

Figure Lengend Snippet: UCHL5 is highly expressed in hepatocellular carcinoma, and the prognosis of patients with high expression is poor . ( A ) Expression of the UCHL5 was validated in 369 HCC tissues and 160 normal tissues with GEPIA. Tumor tissue is shown in red, and normal tissue is shown in gray; ( B ) The AUC value of a single UCHL5 gene in hepatocellular carcinoma; ( C , D ) The mRNA and protein expression of UCHL5 in hepatocellular carcinoma and adjacent normal tissues were analyzed by using RT-qPCR and immunohistochemistry; ( E ) Prognosis of patients with different UCHL5 expression levels; ( F ) The expression of UCHL5 in 14 cases of hepatocellular carcinoma and adjacent tissues was detected by Western blot; ( G , H ) The expression of UCHL5 mRNA and protein expression in human normal hepatocytes (HL-7702) and six hepatocellular carcinoma cells (BEL7402, PLC/PRF/5, Huh-7, HepG2, HepG3B and SNU449). Data represent the mean ± SD of three independent experiments. GAPDH as internal parameter. (* p < 0.05, ** p < 0.01 and *** p < 0.001)

Article Snippet: Negative control and UCHL5 -shRNA lentiviral particles were bought from Genechem to create a cell line that would permanently silence the UCHL5 gene (Shanghai, China).

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

Journal: BMC Cancer

Article Title: UCHL5 promotes hepatocellular carcinoma progression by promoting glycolysis through activating Wnt/β-catenin pathway

doi: 10.1186/s12885-023-11317-z

Figure Lengend Snippet: UCHL5 promotes the proliferation and metastasis of HCC cells in vitro . ( A , B ) ShUCHL5 or sh-control were transfected into HepG2 and Hep3B cells. The mRNA and protein levels of UCHL5 were examined by RT-qPCR and western blot; ( C , E ) The effect of UCHL5 on HCC cell proliferation was determined by CCK-8 ( C ) and colony formation ( E ); ( D ) Cell scratch assay to determine the effect of UCHL5 on the migratory ability of HCC cells; ( F ) The effect of UCHL5 on migration (left) and invasion (right) were evaluated by Transwell assays. Data represent the mean ± SD of three independent experiments. GAPDH as internal parameter. (* p < 0.05, ** p < 0.01 and *** p < 0.001)

Article Snippet: Negative control and UCHL5 -shRNA lentiviral particles were bought from Genechem to create a cell line that would permanently silence the UCHL5 gene (Shanghai, China).

Techniques: In Vitro, Control, Transfection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Wound Healing Assay, Migration

Journal: BMC Cancer

Article Title: UCHL5 promotes hepatocellular carcinoma progression by promoting glycolysis through activating Wnt/β-catenin pathway

doi: 10.1186/s12885-023-11317-z

Figure Lengend Snippet: UCHL5 promotes the glycolysis of HCC cells . ( A ) UCHL5 co-expression genes in TCGA were enriched in KEGG pathways; ( B , C , D ) Detection of glucose consumption ( B ), lactate production ( C ), intracellular ATP level ( D ) and ECAR( E ) in transfected cells; ( F ) The mRNA levels of GLUT1, PKM2, HK2 , LDHA , LDHB , and PDK1 were examined by RT-qPCR after transfected with shUCHL5 or sh-control. Data represent the mean ± SD of three independent experiments. (* p < 0.05)

Article Snippet: Negative control and UCHL5 -shRNA lentiviral particles were bought from Genechem to create a cell line that would permanently silence the UCHL5 gene (Shanghai, China).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Control

Journal: BMC Cancer

Article Title: UCHL5 promotes hepatocellular carcinoma progression by promoting glycolysis through activating Wnt/β-catenin pathway

doi: 10.1186/s12885-023-11317-z

Figure Lengend Snippet: UCHL5 reduces β-catenin degradation through deubiquitination . ( A ) Immunoprecipitation of UCHL5 or IgG was carried out in HCC cells. The precipitated protein was analyzed by SDS-PAGE, silver staining was carried out, and the differential bands were identified by mass spectrometry (MS); ( B ) Co-IP demonstrated the binding between UCHL5 and β-catenin protein; ( C , E ) The mRNA and protein levels of β-catenin were examined by RT-qPCR ( E ) and western blot ( C ) after knockdown or overexpression of UCHL5 ; ( D ) The expression level of β-catenin in different groups ( UCHL5 high or UCHL5 low) were showed by IHC; ( F ) The protein levels of β-catenin and the ubiquitination level of β-catenin were examined by western blot after overexpression of UCHL5 or addition of MG132; ( G ) The protein levels of β-catenin were examined in different time points after adding CHX by western blot and gel blots quantification; ( H , I ) The TOP/FOP fold change after knockdown or overexpression of UCHL5 ; ( J , K , L ) The mRNA and protein levels of cyclinD1, c-Myc, VEGF and survivin were examined by RT-qPCR ( J , K ) and western blot ( L ) after knockdown or overexpression of UCHL5 . Data represent the mean ± SD of three independent experiments. GAPDH as internal parameter. (* p < 0.05)

Article Snippet: Negative control and UCHL5 -shRNA lentiviral particles were bought from Genechem to create a cell line that would permanently silence the UCHL5 gene (Shanghai, China).

Techniques: Immunoprecipitation, SDS Page, Silver Staining, Mass Spectrometry, Co-Immunoprecipitation Assay, Binding Assay, Quantitative RT-PCR, Western Blot, Knockdown, Over Expression, Expressing, Ubiquitin Proteomics

Journal: BMC Cancer

Article Title: UCHL5 promotes hepatocellular carcinoma progression by promoting glycolysis through activating Wnt/β-catenin pathway

doi: 10.1186/s12885-023-11317-z

Figure Lengend Snippet: UCHL5 promotes HCC progression through β-catenin. After changing the expression of UCHL5 , β-catenin or adding IWP-4 . ( A , C ) The change of HCC cell proliferation was determined by CCK-8 ( A ) and colony formation ( C ) in different time points; ( B ) Cell scratch assay to determine the migration ability of HCC cells; ( D ) The change of migration and invasion ability was evaluated byTranswell assays; ( E , F , G ) Detection of glucose consumption ( E ), lactate production ( F ) and intracellular ATP level by RT-qPCR ( G ); The mRNA levels of GLUT1, PKM2, HK2 , and LDHA were examined by RT-qPCR. Data represent the mean ± SD of three independent experiments. (* p < 0.05)

Article Snippet: Negative control and UCHL5 -shRNA lentiviral particles were bought from Genechem to create a cell line that would permanently silence the UCHL5 gene (Shanghai, China).

Techniques: Expressing, CCK-8 Assay, Wound Healing Assay, Migration, Quantitative RT-PCR

Journal: BMC Cancer

Article Title: UCHL5 promotes hepatocellular carcinoma progression by promoting glycolysis through activating Wnt/β-catenin pathway

doi: 10.1186/s12885-023-11317-z

Figure Lengend Snippet: UCHL5 promotes the proliferation and metastasis of HCC cells in vivo . ( A ) Images of xenograft tumors derived from transfected cells in nude mice; ( B ) Tumor volume curves were summarized in the line chart. The average tumor volume was expressed as the mean standard deviation of 5 mice; ( C ) The tumor weight was assessed in different groups; ( D ) The expression level of UCHL5 , β-catenin, KI67, and PCNA in different groups were shown by IHC. ( E ) Representative images of the tail vein-injected mouse models. ( F ) The lung foci number was evaluated. Data represent the mean ± SD of three independent experiments. (** p < 0.01)

Article Snippet: Negative control and UCHL5 -shRNA lentiviral particles were bought from Genechem to create a cell line that would permanently silence the UCHL5 gene (Shanghai, China).

Techniques: In Vivo, Derivative Assay, Transfection, Standard Deviation, Expressing, Injection

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Schlafen 12 Interaction with SerpinB12 and Deubiquitylases Drives Human Enterocyte Differentiation

doi: 10.1159/000492019

Figure Lengend Snippet: Schlafen 12 acts by modulating deubuiquitylase activity. (a) Caco-2 cells transfected with non-targeting siRNA5 (siNT5) or a combination of both USP14 (siUSP14) and UCHL5 (siUCHL5) siRNA followed by transduction with control (Ad-CMV) or SLFN12 (Ad-SLFN12) viruses for 72 hours, and SI expression was assessed (n=6,*p<0.05). (b,c) Caco-2 cells were transduced with Ad-CMV or Ad-SLFN12 for 72 hours and USP14 and UCHL5 expression was measured (n=6 each,*p<0.05). (d,e,f) Caco-2 cells were transfected with either nontargeting siRNA NT5 or combined siRNA to UCHL5 and USP15 along with either an empty vector adenovirus (Ad-V) or adenovirus encoding SLFN12 (AdSLFN12). Western blots were performed for sucraseisomaltase (d), USP14 (e), and UCHL5 (f). (g,h) Caco-2 cells were transduced with Ad-CMV or Ad-SERPB12 and USP14 and UCHL5 expression measured. (i) Caco-2 cell lysates were immunoprecipitated with monoclonal USP14 or monoclonal SerpinB12 antibodies and immunoblotted with polyclonal SerpinB12 antibody. We immunoprecipitated Serpin B12 directly as a control to confirm the correct apparent molecular weight of the co-precipitating Serpin B12. (j) Incubation with recombinant human Serpin B12 stimulated deubuiquitylase activity vs. USP14 alone (n=4,*p<0.05 to substrate (sub) or SERPB12 alone; # p<0.05 vs. USP14 alone). (k) Incubation with recombinant human Serpin B12 did not affect the UCHL5 deubuiquitylase activity vs. UCHL5 alone (n=4,*p<0.05 to substrate (Sub) or Serpin B12). All statistics are by two-sided t test with Bonferroni corrections for multiple comparisons.

Article Snippet: Human recombinant USP14 (Cat# E544) and human recombinant UCHL5 (Cat#E327–025) were from Boston Biochem Inc (Cambridge, MA) and human recombinant SerpinB12 (C6-His, Catalog# CI96) was from Novoprotein (Summit, NJ).

Techniques: Activity Assay, Transfection, Transduction, Control, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation, Molecular Weight, Incubation, Recombinant

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Schlafen 12 Interaction with SerpinB12 and Deubiquitylases Drives Human Enterocyte Differentiation

doi: 10.1159/000492019

Figure Lengend Snippet: Schlafen 12 induces CDX2 transcription factor and its effects are blocked by reducing USP14 and UCHL5 deubiquitylase. (a,b) Caco-2 cells were transduced with Ad-CMV or Ad-SLFN12 for 72 hours and CDX2 or CDX4 protein was measured (n=5 for each, p>0.05). (c) Caco-2 cells were transduced with Ad-CMV or Ad-SLFN12 for 72 hours and CDX2 mRNA was assessed (n=5, p>0.05). (d) Caco-2 cells transfected with non-targeting siRNA5 (siNT5) or a combination of both USP14 (siUSP14) and UCHL5 (siUCHL5) siRNA followed by transduction with Ad-CMV or Ad-SLFN12 for 72 hours, lysis, and western blotting using CDX2 or GAPDH antibodies (n=6,*p<0.05). (e) ≈50% reduction of CDX2 by siRNA (siCDX2), data not shown prevents the induction of sucrase-isomaltase promoter activity by cotransfection with a SLFN12 plasmid (SI-P+SLFN12) in comparison to sucrase-isomaltase promoter activity after cotransfection with an empty vector control plasmid (P). In contrast, SI-P+SLFN12 induces sucrase-isomaltase promoter activity after transfection with non-targeting siRNA (siNT5). (n=6, *p<0.05). All statistics are by two-sided t test with Bonferroni corrections for multiple comparisons.

Article Snippet: Human recombinant USP14 (Cat# E544) and human recombinant UCHL5 (Cat#E327–025) were from Boston Biochem Inc (Cambridge, MA) and human recombinant SerpinB12 (C6-His, Catalog# CI96) was from Novoprotein (Summit, NJ).

Techniques: Transduction, Transfection, Lysis, Western Blot, Activity Assay, Cotransfection, Plasmid Preparation, Comparison, Control

Journal: Biochimica et Biophysica Acta. Molecular Cell Research

Article Title: A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate ☆

doi: 10.1016/j.bbamcr.2012.05.006

Figure Lengend Snippet: Ubiquitylation of DUB Chip. A. Individual, identical protein microarrays were ubiquitylated with five different E3s (individually as listed), a mixture of three labeled “mix” (Praja1, Carp2, and Murf1), or using reaction mixture lacking an E3 (“No E3”). All substrates were printed in triplicate, and all conditions were tested on two separate (identical) arrays. One of the two was visualized using antibody capable of seeing monoubiquitylation events (left column of arrays labeled “Ab”); the other using polyubiquitin-specific TUBEs (right column of arrays labeled “TUBEs”). B. E3Lite assay (LifeSensors, Inc.) data (signal versus time) showing all E3s used in (A) were capable of forming polyubiquitin chains (either free or E3-conjugated). C. Data from ubiquitylation reactions (A) visually organized by E3 (columns) and substrate (rows). The column labeled “TUBEs” represents the array ubiquitylated with E3 mix and visualized with TUBEs; all other columns represent data from arrays visualized with antibody. The identities of substrates by row are: 1. USP20, 2. USP51c, 3. USP5, 4. Ataxin3, 5. USP7c, 6. AMSH, 7. Otubain1, 8. JOSD1, 9. UCHL3, 10. USP8c, 11. UCHL5, 12. SENP6c, 13. SENP1c, 14. USP21, 15. Otubain2, 16. Ataxin3-like, 17. USP33c, 18. USP15, 19. USP28, 20. sseL, 21. USP18, 22. DEN1, 23. PLPro, 24. SENP2, 25. USP34c, 26. USP14, 27. USP8, 28. hSTAM1, 29. USP2c, 30. USP7, 31. BAP1, 32. JOSD2, 33. YOD1, 34. UCHL1, 35. USP21c, 36. Ubiquitin, 37. Ulp1c, 38. PLP2, 39. USP4.

Article Snippet: Chicken IgY antibodies to UCHL5 and JOSD1 (LifeSensors, Inc.) were used at 1:1000 dilution in PBST and visualized with horseradish peroxidase-conjugated rabbit anti-chicken IgY (Rockland #803-4302) diluted 1:2000 in PBST.

Techniques: Labeling, Ubiquitin Proteomics

Journal: Biochimica et Biophysica Acta. Molecular Cell Research

Article Title: A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate ☆

doi: 10.1016/j.bbamcr.2012.05.006

Figure Lengend Snippet: Deubiquitylation of DUB Chip. A. Sixteen identical protein microarrays were all ubiquitylated with E3 mix (Praja1, Carp2, and Murf1). After washing away soluble ubiquitylation machinery, individual DUBs (as listed, including mock-treated arrays labeled “No DUB”) were applied to separate arrays, which were then visualized by antibody capable of seeing monoubiquitylation events. Loss of signal indicates putative substrates of the DUB tested. B. Array data from (A) visually organized by DUB tested (columns) and ubiquitylated substrates (rows). The identities of substrates by row are: 1. USP20, 2. USP51c, 3. USP5, 4. Ataxin3, 5. USP7c, 6. AMSH, 7. Otubain1, 8. JOSD1, 9. UCHL3, 10. USP8c, 11. UCHL5, 12. USP21, 13. Otubain2, 14. Ataxin3-like, 15. USP33c, 16. USP15, 17. USP28, 18. sseL, 19. DEN1, 20. PLPro, 21. USP14, 22. USP8, 23. hSTAM1, 24. USP2c, 25. USP7, 26. BAP1, 27. YOD1, 28. UCHL1, 29. USP21c, 30. Ubiquitin, 31. Ulp1c, 32. PLP2, 33. USP4. C. Experiment identical to that described in (A), except that the array was visualized using polyubiquitin-specific TUBEs. D. Array data from (C) visually organized by DUB tested (columns) and ubiquitylated substrates (rows, as above).

Article Snippet: Chicken IgY antibodies to UCHL5 and JOSD1 (LifeSensors, Inc.) were used at 1:1000 dilution in PBST and visualized with horseradish peroxidase-conjugated rabbit anti-chicken IgY (Rockland #803-4302) diluted 1:2000 in PBST.

Techniques: Labeling, Ubiquitin Proteomics

Journal: Biochimica et Biophysica Acta. Molecular Cell Research

Article Title: A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate ☆

doi: 10.1016/j.bbamcr.2012.05.006

Figure Lengend Snippet: E3 auto(poly)ubiquitylation and DUB monoubiquitylation in gel. A. Ubiquitylation reactions with Murf1 (shown by array to monoubiquitylate DUB substrates) as the E3 were run in the presence of various DUBs. Reactions were SDS PAGE separated, transferred to nitrocellulose, and probed with TUBEs to observe any high molecular weight polyubiquitylation. Most reactions with DUBs displayed enhanced smearing characteristic of polyubiquitylation, compared to the reaction containing no DUB (first lane). B. Antibody to UCHL5 was used to probe separated reaction components from complete ubiquitylation reactions including Praja1 as the E3 (shown by array to be capable of DUB polyubiquitylation) and UCHL5 as substrate (column labeled (+)). Inclusion of all components except Praja1 (column labeled “-E3”) showed that the observed 8 kD shift (indicated by arrow) above the predominant form of the substrate was ubiquitylation dependent. Similarity of this lane to one containing only UCHL5 protein substrate (column labeled “Pr”) confirms that the antibody was not cross reacting to any ubiquitylation machinery (e.g., ubiquitin or charged E2). C. Identical to Fig. 5 B except that blot was visualized with anti-JOSD1 antibody, and JOSD1 was the substrate included in the reactions (or protein alone lane). D. Ubiquitin antibody (VU1) was used to visualize (via immunoblot) soluble components of in vitro ubiquitylation reactions in which E3 was pre-immobilized to the surface of the well (and therefore not present in the gel/blot) and DUB omitted (negative control, lane 1); where all components were soluble including UCHL5 as substrate (positive control, lane 2); and where UCHL5 was included but the E3 (Murf2) was again pre-immobilized to the well surface (lane 3). Arrow indicates expected size of monoubiquitylated UCHL5.

Article Snippet: Chicken IgY antibodies to UCHL5 and JOSD1 (LifeSensors, Inc.) were used at 1:1000 dilution in PBST and visualized with horseradish peroxidase-conjugated rabbit anti-chicken IgY (Rockland #803-4302) diluted 1:2000 in PBST.

Techniques: SDS Page, High Molecular Weight, Labeling, Ubiquitin Proteomics, Western Blot, In Vitro, Negative Control, Positive Control

Journal: Biochimica et Biophysica Acta. Molecular Cell Research

Article Title: A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate ☆

doi: 10.1016/j.bbamcr.2012.05.006

Figure Lengend Snippet: Deubiquitylation of soluble ubiquitylated substrates. In vitro ubiquitylation reactions with Praja1 as the E3 were performed in the presence of either JOSD1 or UCHL5 as the intended substrate. After 30 min, a second deubiquitylase enzyme (either USP2c, USP21c, PLP2, or Ataxin3, as indicated) was added to the reactions which were then incubated for another hour. Shown are immunoblots of the separated reaction components probed with primary antibody against the corresponding intended substrate (JOSD1 or UCHL5).

Article Snippet: Chicken IgY antibodies to UCHL5 and JOSD1 (LifeSensors, Inc.) were used at 1:1000 dilution in PBST and visualized with horseradish peroxidase-conjugated rabbit anti-chicken IgY (Rockland #803-4302) diluted 1:2000 in PBST.

Techniques: In Vitro, Incubation, Western Blot

Journal: bioRxiv

Article Title: Impairment of proteasome-associated deubiquitinating enzyme Uchl5/UBH-4 affects autophagy

doi: 10.1101/2024.04.04.588054

Figure Lengend Snippet: A. Schematic representation of the GFP-LC3-RFP-LC3ΔG fluorescent probe . B. GFP-LC3-RFP-LC3ΔG HeLa cells treated with control, Uchl5 or USsp14 siRNA and collected 48h post-transfection. Expression of Uchl5 or Usp14 mRNA was measured with qPCR. Graph shows the percentage change in the mRNA levels compared to control (set as 100%). C. Whole cell extracts (48h post-transfection) were analyzed by SDS-PAGE and immunoblotted against Uchl5, Usp14 and HSC70. The graphs (on right panel) show average fold change in levels of Uchl5 and Usp14 normalized against HSC70. Results are the mean of quantifications from three (Uchl5) or one (Usp14) independent experiment. Error bars, SEM, *p<0,05 compared to the control (set as 1).

Article Snippet: For siRNA experiments, FlexiTube GeneSolution for uchl5 and usp14 (QIAGEN) and AllStars Negative Control siRNA (QIAGEN) were used with HiPerFect transfection Reagent (QIAGEN).

Techniques: Transfection, Expressing, SDS Page

Journal: bioRxiv

Article Title: Impairment of proteasome-associated deubiquitinating enzyme Uchl5/UBH-4 affects autophagy

doi: 10.1101/2024.04.04.588054

Figure Lengend Snippet: A. Fluorescence confocal images of control, Uchl5 or Usp14 siRNA treated GFP-LC3-RFP-LC3ΔG HeLa cells after 48h post-transfection. Insets show enlarged view of the indicated areas. Magenta arrows point to some of the GFP-LC3 puncta. Scale bar, 20 µm. The right upper graph shows quantification of relative fold change in the number of GFP-LC3 puncta per image (Control set at 1). The right lower graph shows quantification of the relative fold change in the ratio of GFP to RFP per image (Control set at 1). Results are from three independent experiments (total 15-17 images were analyzed). Error bars, SEM, *p<0,05, ***p<0,001 compared to control. B and C . GFP-LC3-RFP-LC3ΔG HeLa cells treated with control, Uchl5 or Usp14 siRNA for 48h. Whole cell extracts were analyzed by SDS-PAGE and immunoblotted against LC3, p62 and HSC70. The graphs (on right panels) show average fold change in levels of LC3 (B) and p62 (C) normalized against HSC70. Results are the mean of quantifications from 3-4 independent experiments. Error bars, SEM, *p<0,05 compared to the control (set as 1).

Article Snippet: For siRNA experiments, FlexiTube GeneSolution for uchl5 and usp14 (QIAGEN) and AllStars Negative Control siRNA (QIAGEN) were used with HiPerFect transfection Reagent (QIAGEN).

Techniques: Fluorescence, Transfection, SDS Page

Journal: bioRxiv

Article Title: Impairment of proteasome-associated deubiquitinating enzyme Uchl5/UBH-4 affects autophagy

doi: 10.1101/2024.04.04.588054

Figure Lengend Snippet: A Graphs show the quantification of the number of puncta positive for GFP and mCherry-only in the intestinal cells, hypodermal seam cells and the pharynx. All animals, except the ones labelled second generation, were exposed to RNAi from L1 larval stage to day 1 of adulthood (First generation). The animals and their progeny were further continuously exposed to RNAi, and the day 1 of adulthood of the progeny is here labelled as second generation. Results are from three independent experiments. Puncta were counted from a total of 20-30 individual corresponding cells. Error bars, SEM, **p<0,01, ***p<0,001 compared to control. B. Fluorescence confocal micrographs of control, ubh-4 or usp14 RNAi-treated mCherry::GFP::LGG-1 animals showing the pharynx at day 1 of adulthood (First generation). Graphs show the quantification of the number of puncta positive for GFP and mCherry-only. Results are from three independent experiments. Puncta were counted from a total of 10-12 pharynges from 10-12 animals. Error bars, SEM.

Article Snippet: For siRNA experiments, FlexiTube GeneSolution for uchl5 and usp14 (QIAGEN) and AllStars Negative Control siRNA (QIAGEN) were used with HiPerFect transfection Reagent (QIAGEN).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Impairment of proteasome-associated deubiquitinating enzyme Uchl5/UBH-4 affects autophagy

doi: 10.1101/2024.04.04.588054

Figure Lengend Snippet: Fluorescence confocal micrographs of control, ubh-4 or usp14 RNAi-treated mCherry::GFP::LGG-1 animals showing hypodermal seam cells (A) and, the pharynx (B). Graphs show the quantification of the number of puncta positive for GFP and mCherry-only. Results are from five independent (for hypodermal seam cells) or three independent (for pharynx (second generation)) experiments. Puncta were counted from a total of 20-25 pharynges from 20-25 animals and 25-30 hypodermal seam cells from 20-25 animals. Error bars, SEM, **p<0,01 and ***p<0,001 compared to control.

Article Snippet: For siRNA experiments, FlexiTube GeneSolution for uchl5 and usp14 (QIAGEN) and AllStars Negative Control siRNA (QIAGEN) were used with HiPerFect transfection Reagent (QIAGEN).

Techniques: Fluorescence