Journal: PLoS ONE

Article Title: USP7 and TDP-43: Pleiotropic Regulation of Cryptochrome Protein Stability Paces the Oscillation of the Mammalian Circadian Clock

doi: 10.1371/journal.pone.0154263

Figure Lengend Snippet: A. Silver staining image of proteins co-purified with FLAG-His-Myc-CRY1 (FHM-CRY1) or FHM-CRY2. NIH3T3 cells expressing FHM-CRY1 or FHM-CRY2 were treated with 10 μM MG132 for 6 hours and lysed with IP Buffer. Cell lysates were subjected to immunoprecipitation using anti-FLAG-M2 agarose beads. FH-LacZ expressed in NIH3T3 cells was used as a control. B . The numbers of proteins co-purified with FHM-CRY1 or FHM-CRY2. Proteins co-purified with FH-LacZ were eliminated from the list of CRY1 and CRY2 interacting proteins. Proteins detected in both CRY1 and CRY2 samples with high MS scores were listed in . C. Interaction of USP7 with CRY2 protein. NIH3T3 cells expressing FLAG-CRY2 and/or Myc-USP7 were cultured in the presence of 10 μM MG132 for 6 hours and lysed with IP Buffer. The cell lysates were subjected to immunoprecipitation using anti-FLAG, anti-Myc antibody or normal mouse IgG (negative control) as precipitating antibodies.

Article Snippet: USP7 specific inhibitor HBX 41108 was purchased from Boston Biochem and solved in DMSO, which was used as a vehicle control of the inhibitor treatment.

Techniques: Silver Staining, Purification, Expressing, Immunoprecipitation, Control, Cell Culture, Negative Control

Journal: PLoS ONE

Article Title: USP7 and TDP-43: Pleiotropic Regulation of Cryptochrome Protein Stability Paces the Oscillation of the Mammalian Circadian Clock

doi: 10.1371/journal.pone.0154263

Figure Lengend Snippet: A . In vitro ubiquitination assay. HEK293T/17 cells were transfected with the expression vector of FLAG-CRY2. Forty-two hours after the transfection, the cells were cultured in the presence of 10 μM MG132 for 6 hours and then harvested. FLAG-CRY2 purified from the cell lysate with anti-FLAG M2 agarose beads was incubated with or without a recombinant protein, full-length USP7 or a catalytic domain of USP2 (USP2 CD), for 30 min at 37°C. Recombinant USP2 catalytic domain was used as a positive control . B . In vivo deubiquitination assay in HEK293T/17 cells. The cells were transfected with indicated expression vectors. Forty-two hours after the transfection, the cells were cultured in the presence of 10 μM MG132 for 6 hours and then lysed with IP Buffer. FLAG-CRY2 was purified with anti-FLAG M2 agarose beads, followed by western blotting analysis with anti-CRY2 antibody. An inactive mutant of USP7 (USP7-C223A) was used for a negative control. C. Effect of USP7-specific inhibitor on CRY up-shifted bands. NIH3T3 cells were transfected with the expression vector for Myc-CRY1 or Myc-CRY2. Forty-two hours after the transfection, the cells were cultured in the presence of 20 μM HBX 41108 for 6 hours. The smear bands of Myc-CRY1 or Myc-CRY2 were quantified (means + SEM, n = 3, **: p < 0.01 by Student’s t -test).

Article Snippet: USP7 specific inhibitor HBX 41108 was purchased from Boston Biochem and solved in DMSO, which was used as a vehicle control of the inhibitor treatment.

Techniques: In Vitro, Ubiquitin Proteomics, Transfection, Expressing, Plasmid Preparation, Cell Culture, Purification, Incubation, Recombinant, Positive Control, In Vivo, Western Blot, Mutagenesis, Negative Control

Journal: PLoS ONE

Article Title: USP7 and TDP-43: Pleiotropic Regulation of Cryptochrome Protein Stability Paces the Oscillation of the Mammalian Circadian Clock

doi: 10.1371/journal.pone.0154263

Figure Lengend Snippet: A. Effect of USP7 expression on CRY protein levels. HEK293T/17 cells were transfected with indicated expression vectors. Forty-eight hours after the transfection, the cells were lysed with SDS-PAGE sample buffer, and the cell lysate was analyzed by western blotting. GFP was used as a control. Quantified data are shown by means + SEM (n = 3, **: p < 0.01 by Student’s t -test). B. Effect of USP7 overexpression on CRY-LUC protein stability. HEK293T/17 cells were transfected with expression vectors for CRY-LUC and Myc-USP7 (or empty control), and cultured for 48 hours. The culture medium was changed to the recording medium containing 0.1 mg/ml cycloheximide. Bioluminescence signals were recorded continuously at 10-min intervals, and the signal was normalized to the value at time 0. Half-lives of CRY1-LUC and CRY2-LUC were calculated by fitting an exponential decay curve to bioluminescence signals, and are shown as means + SEM (n = 3, **: p < 0.01 by Student’s t -test). C. USP7 inhibitor treatment of NIH3T3 cells decreased Myc-CRY1 expression. NIH3T3 cells were transfected with Myc-CRY1 and GFP expression vectors. Forty-two hours after the transfection, the cell were cultured with 10 or 20 μM HBX 41108 for 6 hours. Quantified data are shown as means + SEM (n = 3). D. Usp7 knockdown decreased the starting levels of CRYs-LUC. The decay of the bioluminescence signals was recorded as described in B . The starting levels of CRYs-LUC bioluminescence signals are shown as means + SEM (n = 4, **: p < 0.01 by Student’s t -test). E. Usp7 knockdown decreased CRYs-LUC stability. The bioluminescence signals normalized to the value at time 0 and the half-lives of CRYs-LUC are shown as means + SEM (n = 4, **: p < 0.01 by Tukey’s test or Student’s t -test). F. Effect of USP7 overexpression on CRY2-LUC protein stability in Fbxl3 knockdown cells. HEK293T/17 cells were transfected with indicated plasmid vectors and cultured for 72 hours. The decay of the bioluminescence signals was recorded as described in B . Quantified data are shown as means + SEM (n = 3, *: p < 0.05 by Student’s t -test).

Article Snippet: USP7 specific inhibitor HBX 41108 was purchased from Boston Biochem and solved in DMSO, which was used as a vehicle control of the inhibitor treatment.

Techniques: Expressing, Transfection, SDS Page, Western Blot, Control, Over Expression, Cell Culture, Knockdown, Plasmid Preparation

Journal: PLoS ONE

Article Title: USP7 and TDP-43: Pleiotropic Regulation of Cryptochrome Protein Stability Paces the Oscillation of the Mammalian Circadian Clock

doi: 10.1371/journal.pone.0154263

Figure Lengend Snippet: A. NIH3T3 cells were transfected with a luciferase reporter vector, Bmal1 us0.3-luc, and USP7 expression vector. Twenty-four hours after the transfection, the cellular rhythms were synchronized by 30-min treatment with 0.1 μM dexamethasone (Dex). The culture medium was changed to the recording medium, and bioluminescence signals were recorded continuously. The calculated circadian periods are shown as means + SEM (n = 4, *: p < 0.05 by Student’s t -test). B. Cellular rhythms in Usp7 knockdown cells. Bioluminescence rhythms of Bmal1 -luc reporter were recorded as described in A . The calculated circadian periods are shown as means + SEM (n = 4, **: p < 0.01 by Tukey’s test).

Article Snippet: USP7 specific inhibitor HBX 41108 was purchased from Boston Biochem and solved in DMSO, which was used as a vehicle control of the inhibitor treatment.

Techniques: Transfection, Luciferase, Plasmid Preparation, Expressing, Knockdown

Journal: Autophagy

Article Title: Hepatitis B virus–triggered autophagy targets TNFRSF10B/death receptor 5 for degradation to limit TNFSF10/TRAIL response

doi: 10.1080/15548627.2016.1239002

Figure Lengend Snippet: HBx downregulates the expression of TNFRSF10B in HBV-infected cells. (A) TNFRSF10B expression levels in HepG-X and HepG-M cells were analyzed by semi-quantitative RT-PCR and immunoblot assay. (B) TNFRSF10B expression at the indicated time points after transfection with the control or HBx plasmid in HepG2 cells was analyzed by immunoblot assay. A representative immunoblot and quantification of the TNFRSF10B signal are shown. (C) TNFRSF10B expression levels in L02-X and L02-M cells were analyzed by semi-quantitative RT-PCR and immunoblot assay. (D) TNFRSF10B expression at the indicated time points after transfection of L02 cells with the control or HBx plasmid was analyzed by immunoblot assay. Quantification of the TNFRSF10B signal is shown. (E) Expression levels of TNFRSF10B in HepG2 and HepG2.2.15 cells were analyzed by semi-quantitative RT-PCR and immunoblot assay. (F) TNFRSF10B expression at the indicated time points after transfection in HepG2 cells with the control or HBV1.2mer plasmid was analyzed by immunoblot assay. A representative immunoblot and quantification of the TNFRSF10B signal are shown. (G) Representative immunocytochemical images of TNFRSF10B expression in HepG2 cells transiently transfected with the HBx plasmid. Cells transfected with the HBx-Flag or pcDNA3.1 vector were stained with anti-Flag (red) and anti-TNFRSF10B (green) antibodies. Among the HBx-positive cells, the percentage of cells showing reduced TNFRSF10B expression compared to control cells is indicated. (H) TNFRSF10B expression levels on the surface of HBx-expressing cells, HepG2.2.15 cells, and the corresponding control cells were analyzed by flow cytometry. Relative TNFRSF10B expression on the cell surface was calculated as the percentage of mean fluorescence intensity (MFI) as described in the Materials and Methods. All data are mean ± SD of 3 independent experiments. P-values were obtained by Student t test (*P < 0.05).

Article Snippet: Antibodies against HBx (BioVendor, RD981038100), HA (Sigma-Aldrich, H6908), Flag M2 (Sigma-Aldrich, F1804), LC3B (Abcam, ab51520), and BECN1 (Atlas Antibodies, HPA028949) were also used.

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Fluorescence

Journal: Autophagy

Article Title: Hepatitis B virus–triggered autophagy targets TNFRSF10B/death receptor 5 for degradation to limit TNFSF10/TRAIL response

doi: 10.1080/15548627.2016.1239002

Figure Lengend Snippet: HBx promotes complete autophagy. Representative immunofluorescence images of autophagic cells (left) and their quantification (right). (A) HepG2 cells were cotransfected with the indicated plasmids and the tfLC3B plasmid. Cells with both red and green fluorescent LC3B dots (autophagosomes) and those with only red fluorescent LC3B dots (autolysosomes) were quantified as described in Materials and Methods. (B, C) HepG-X (B) or HepG2.2.15 (C) cells were transfected with the tfLC3B plasmid, and the formation of autophagosomes and autolysosomes was compared in these and the corresponding control cells as in (A). (D, E) Endogenous levels of LC3B and SQSTM1 in HepG2 (D) and L02 (E) cells were analyzed by immunoblot assay at the indicated time points after transfection with the HBx plasmid or HBV1.2mer. (F) Endogenous levels of LC3B and SQSTM1 in HepG-X, L02-X, and HepG2.2.15 cells were compared with those in the corresponding control cells. All data are mean ± SD of 3 independent experiments. P-values were obtained by Student t test (*P < 0.05).

Article Snippet: Antibodies against HBx (BioVendor, RD981038100), HA (Sigma-Aldrich, H6908), Flag M2 (Sigma-Aldrich, F1804), LC3B (Abcam, ab51520), and BECN1 (Atlas Antibodies, HPA028949) were also used.

Techniques: Immunofluorescence, Plasmid Preparation, Transfection, Western Blot

Journal: Autophagy

Article Title: Hepatitis B virus–triggered autophagy targets TNFRSF10B/death receptor 5 for degradation to limit TNFSF10/TRAIL response

doi: 10.1080/15548627.2016.1239002

Figure Lengend Snippet: HBx-induced autophagy is responsible for TNFRSF10B degradation and downregulation of its cell surface expression. TNFRSF10B expression levels in HepG-X (A) and HepG2.2.15 (B) cells were analyzed after treatment with bafilomycin A1 (BAF) for the indicated times. The level of TNFRSF10B in control cells at each time point was set to 100%. (C) TNFRSF10B expression levels in HepG2 cells transfected with the HBx plasmid, and in HepG-X and HepG2.2.15 cells were analyzed after LC3B knockdown by siRNA. (D, E) HepG-X cells were transfected with the tfLC3B plasmid and treated with DMSO or 3-MA for 16 h (D), or cultured in normal or starvation (Earle's balanced salt solution, EBSS) medium for 16 h (E). Representative immunofluorescence images of autophagy detected by the presence of RFP-LC3B dots. N indicates the nucleus. Under the same conditions, the expression levels of TNFRSF10B and SQSTM1 were analyzed by immunoblotting. (F) Expression levels of TNFRSF10B on the surface of HepG-X and HepG2.2.15 cells were analyzed by flow cytometry after LC3B knockdown by siRNA or after 3-MA treatment. The relative expression of TNFRSF10B on the cell surface was calculated as the percentage of mean fluorescence intensity (MFI) as described in the Materials and Methods. Data are mean ± SD of 3 independent experiments. P-values were obtained by Student t test (*P < 0.05).

Article Snippet: Antibodies against HBx (BioVendor, RD981038100), HA (Sigma-Aldrich, H6908), Flag M2 (Sigma-Aldrich, F1804), LC3B (Abcam, ab51520), and BECN1 (Atlas Antibodies, HPA028949) were also used.

Techniques: Expressing, Transfection, Plasmid Preparation, Cell Culture, Immunofluorescence, Western Blot, Flow Cytometry, Fluorescence

Journal: Autophagy

Article Title: Hepatitis B virus–triggered autophagy targets TNFRSF10B/death receptor 5 for degradation to limit TNFSF10/TRAIL response

doi: 10.1080/15548627.2016.1239002

Figure Lengend Snippet: HBx facilitates the recruitment of TNFRSF10B to the autophagy machinery through ternary complex formation between HBx, TNFRSF10B, and LC3B. (A) Analysis of the interactions among LC3B, TNFRSF10B, and HBx in HBx-expressing cells by immunoprecipitation. At 48 h after transfection with the HBx-HA construct, cell lysates were immunoprecipitated with anti-LC3B antibody and proteins were detected by immunoblot assay. (B, C) Immunoprecipitation with anti-TNFRSF10B and anti-HA antibodies. (D) Analysis of the interaction between HBx and TNFRSF10B in LC3B knockdown cells by immunoprecipitation with anti-HA antibody. (E–G) Analysis of the interactions among LC3B, TNFRSF10B, and HBx in vitro by using a GST affinity isolation assay. GST or GST-HBx immobilized on glutathione resin was incubated with L02 cell lysates (E), purified recombinant TNFRSF10B (F), or purified recombinant LC3B (G). An aliquot of each incubation mixture was analyzed by immunoblotting to examine the input levels of each protein. Another aliquot was used for affinity isolation experiments and the bound proteins were detected by immunoblot assay. (H) Analysis of a direct interaction between LC3B and TNFRSF10B in vitro. Recombinant GST or GST-LC3B immobilized on glutathione resin was incubated with purified recombinant TNFRSF10B, and the affinity isolation assay was performed as above. (I) Proposed mechanism of HBx-mediated TNFRSF10B recruitment to the autophagy machinery. HBx activates autophagy and promotes TNFRSF10B recruitment to the autophagy machinery via interaction with LC3B, leading to TNFRSF10B degradation. This effect is blocked by LC3B knockdown, resulting in TNFRSF10B transport to the cell surface.

Article Snippet: Antibodies against HBx (BioVendor, RD981038100), HA (Sigma-Aldrich, H6908), Flag M2 (Sigma-Aldrich, F1804), LC3B (Abcam, ab51520), and BECN1 (Atlas Antibodies, HPA028949) were also used.

Techniques: Expressing, Immunoprecipitation, Transfection, Construct, Western Blot, In Vitro, Isolation, Incubation, Purification, Recombinant

Journal: Autophagy

Article Title: Hepatitis B virus–triggered autophagy targets TNFRSF10B/death receptor 5 for degradation to limit TNFSF10/TRAIL response

doi: 10.1080/15548627.2016.1239002

Figure Lengend Snippet: HBx-induced autophagy plays a cytoprotective role against TNFSF10-mediated cell death. (A) Cell viability and (B) apoptosis in cells stably expressing HBx. Cells were treated with TNFSF10 for 16 h, and MTT assay or flow cytometry analysis was performed as described in the Materials and Methods. (C) CASP activation in HepG-X cells. Cells were treated with TNFSF10 (100 ng/ml) for the indicated times, and immunoblot assay was performed. Pro, procaspase. For CASP3, short and long exposures are shown to visualize the procaspase and cleaved (active) forms, respectively. (D, E) Cell viability (left) and apoptosis (right) after LC3B knockdown by siRNA (D) or 3-MA treatment (E). (F) Caspase activity in HepG-X cells upon LC3B knockdown. All data are mean ± SD of 3 independent experiments. P-values were obtained by ANOVA (*P < 0.05).

Article Snippet: Antibodies against HBx (BioVendor, RD981038100), HA (Sigma-Aldrich, H6908), Flag M2 (Sigma-Aldrich, F1804), LC3B (Abcam, ab51520), and BECN1 (Atlas Antibodies, HPA028949) were also used.

Techniques: Stable Transfection, Expressing, MTT Assay, Flow Cytometry, Activation Assay, Western Blot, Activity Assay